Therapeutic compositions and methods for treating HIV including identification and manipulation of particular domains associated with immunogenicity

ABSTRACT

Therapeutic compositions and methods for treating HIV including identification and manipulation of particular domains associated with immunogenicity.

RELATIONSHIP TO OTHER APPLICATIONS

This is a US national patent application which is a continuation-in-partof three PCT applications, and to the extent allowed by law, claimspriority to and the benefit of the applications below:

U.S. provisional application 61/195,112 filed 4 Oct. 2008; inventors:Phillip Berman, Sara O'Rourke, Becky Schweighardt, William Scott, FarukSinangil, Terri Wrin, titled ‘Use of intrapatient sequence variation toidentify mutations in the HIV envelope glycoprotein that affect bindingof broadly neutralizing antibodies’;

and international application No. PCT/US09/59583 filed 5 Oct. 2009;inventors: Phillip Berman, Sara O'Rourke, William Scott; Titled‘Selection of HIV vaccine antigens by use of intrapatient sequencevariation to identify mutations in the HIV envelope glycoprotein thataffect the binding of broadly neutralizing antibodies’;

and U.S. Provisional application No. 61/253,858 filed 22 Oct. 2009;inventors: Phillip Berman, Sara O'Rourke, William Scott, titled‘Therapeutic compositions that disrupt the hydrogen bonded ringstructure in gp41 and methods for treating HIV’;

and international application PCT/US10/53637; filed 22 Oct. 2010;inventors: Phillip Berman, Sara O'Rourke, William Scott, titled‘Therapeutic compositions that disrupt the hydrogen bonded ringstructure in gp41 and methods for treating HIV’

and to U.S. provisional application 61/258,833 filed 6 Nov. 2009,inventor: Phillip Berman, titled ‘Method to Improve the Immunogenicityof Vaccine Antigens by Modification of Cleavage Sites in HIV1 gp120’

and to International application No. PCT/US2010/055747 filed 5 Nov.2010, inventor: Phillip Berman, titled ‘Method to Improve theImmunogenicity of Vaccine Antigens by Modification of Cleavage Sites inHIV1 gp120’.

STATEMENT OF SUPPORT

This invention was made with support of the Bill and Melinda GatesFoundation and the University of California, Santa Cruz start-up fund.

SEQUENCE LISTING

The information recorded in electronic form (if any) submitted (underRule 13ter if appropriate) with this application is identical to thesequence listing as contained in the application as filed.

FIELD OF THE INVENTION

The Invention (SC2009-449) relates to therapeutic compositions andmethods for treating HIV and other viral diseases and to vaccines forpreventing HIV and other viral diseases. Specifically the inventionrelates to therapeutic applications against HIV infections and tomethods for creation, screening and identification of viral epitopes ofHIV that have therapeutic value.

Additionally the invention (UCSC2008-776) relates to methods use ofintrapatient sequence variation data to identify mutations in the HIVenvelope glycoprotein that affect the binding of broadly neutralizingantibodies.

Additionally the invention (SC2010-117) relates to methods for improvingthe immunogenicity of HIV antigens by mutation of protease cleavagesites in regions important for receptor binding and the binding ofneutralizing antibodies; and therapeutic compositions and vaccines.

BACKGROUND

A major goal in HIV vaccine research is the identification of antigensable to elicit the production of broadly neutralizing antibodies (bNAbs)effective against primary isolates of HIV. The applicant hasinvestigated the molecular features of the HIV-1 envelope glycoproteins,gp160, gp120 and gp41, that confer sensitivity and resistance of virusesto neutralization.

BRIEF DESCRIPTION OF THE INVENTION

Disclosed are therapeutic compositions and methods for treating HIV andother viral diseases and vaccines for preventing HIV and other viraldiseases. These therapeutic compositions contain species andcompositions that have been identified by a novel method for identifyingmutations in envelope proteins, which mutations provide enhancedsensitivity to neutralization of an virus by anti-viral antisera; inparticular neutralization of an HIV virus by anti-HIV antisera. Thisnovel method is disclosed in US provisional application 61/195,112 filed4 Oct. 2008 and also in related International application No.PCT/US09/59583 filed 5 Oct. 2009, both of which are incorporated byreference in their entirety and which disclose novel methods comprisinganalyzing intra-patient HIV-1 virus variation to identify specific aminoacid residues of the HIV-1 envelope glycoproteins, gp160, gp120, andgp41 that affect sensitivity or resistance to broadly neutralizing HIV-1antibodies. Also provided are proteins identified by these methods, thenucleic acids encoding the proteins, and vaccines comprising theproteins and nucleic acids.

Identification of the determinants of sensitivity and resistance tobroadly neutralizing antibodies is a high priority for HIV research.Analysis of the swarm of closely related envelope protein variants inHIV infected individuals revealed a mutation that markedly affectedsensitivity to neutralization by antibodies and antiviral entryinhibitors targeting both gp41 and gp120. This mutation mapped to theC34 helix of gp41 and disrupted an overlooked structural featureconsisting of a ring of hydrogen bonds in the gp41 trimer. This mutationaffects the assembly of the 6 helix bundle required for virus fusion,and alters the conformational equilibrium so as to favor the pre-hairpinintermediate conformation required for the binding of the HIV-1 envmembrane proximal external region (MPER) specific neutralizingantibodies, 2F5 and 4E10, and the antiviral drug, FUZEON. Targetingcooperative interactions that stabilize conformational transitionsprovides new approach to the design of vaccine antigens and antiviralcompounds. Methods for measuring the integrity of the pre-hairpinintermediate conformation include those described by Yang Xu et al.,“Development of a FRET Assay for Monitoring of HIV gp41 Core Disruption”J. Org. Chem. 2007, 72, 6700-6707.

The invention encompasses the use of various compounds used fortherapeutic purposes. These compounds may be contacted with a virus,such as an HIV virus, and interact with and/or bind to one or moreregions on the viral envelope (env) protein or other viral protein orglycoprotein. This interaction thereby (i) exposes one or morepreviously unexposed epitopes which epitope can bind specifically with aneutralizing antibody and/or (ii) limits, inhibits or prevents fusion ofa viral membrane with a cell membrane, thereby inhibiting infection of acell by a virus. Such compounds may be therapeutic compositions, drugs,small molecules or antibodies.

Also disclosed are therapeutic methods that employ compositions such asdrugs and small molecules or antibodies that interact with specificantigens or epitopes or regions of the glycoproteins or polypeptidesdescribed, thereby (i) exposing a previously unexposed epitope whichepitope can bind specifically with a neutralizing antibody and/or (ii)limiting, inhibiting or preventing fusion of a viral membrane with acell membrane, thereby inhibiting infection of a call by a virus. Alsodisclosed are the therapeutic compositions, drugs, small molecules orantibodies used in the above method.

Also disclosed are generic and specific sequences, mutations, antigensand epitopes that may be used therapeutically for the treatment and/orprevention of viral infection such as HIV infection, and vectors,pseudoviruses and other constructs that comprise specific polynucleotidesequences and mutations that encode antigens and epitopes of theinvention.

Also disclosed are therapeutic methods that comprise delivering avaccine to a subject wherein the vaccine may comprise one or moreantibodies or antigens or epitopes of the invention, or polynucleotidesequences or vectors encoding antigens and epitopes of the invention.

Also disclosed are therapeutic methods and therapeutic compositionscomprising drugs such as small molecules that target a specific antigensor epitopes of the invention, thereby limiting, inhibition or preventingfusion of a viral membrane with a cell membrane, thereby inhibitinginfection of a call by a virus.

One particular embodiment is a method for inhibiting the fusion of anHIV virus to a host cell, the method comprising exposing the HIV virusto a drug compound that disrupts the hydrogen-bonded ring structurebetween the N36 and C34 helices of gp41.

Another embodiment is a method for increasing the immunogenicity of HIVenvelope proteins the method comprising exposing the HIV virus to a drugcompound that disrupts the hydrogen bonded ring structure between theN36 and C34 helices of gp41. Methods for measuring the integrity of thehydrogen-bonded ring structure are known in the art and include thosedescribed by Yang Xu et al., “Development of a FRET Assay for Monitoringof HIV gp41 Core Disruption” J. Org. Chem. 2007, 72, 6700-6707. Thedrugs used in these methods may be, for example, antibodies, smallmolecules or peptidomimetics, including, for example, FUZEON, 4E10, 2F5,Q665R, Q655K and Q655E. In some of the methods the drug compoundinteracts with the gp120 fragment of the HIV envelope protein. In someof the methods, the drug compound is an inhibitor of HIV fusion bindingand becomes a more effective inhibitor in the presence of a moleculethat disrupts the disulfide bonded ring structure of gp41.

An important element of the invention is the mechanism by which the drugworks to prevent viral fusion and/or to expose previously hiddenepitopes. In various methods the drug compound disrupts the hydrogenbonded ring structure between the N36 and C34 helices of gp41 andthereby exposes neutralizing epitopes which are recognized by endogenousor exogenous antibodies which then are able to neutralize the virus.

The invention encompasses methods for screening for a drug that preventsor attenuates intracellular membrane fusion, the method comprisingexposing the multimeric coiled coil bundle of the activated fusioncomplex to a drug candidate, wherein disruption of one or more hydrogenbonds of the fusion complex is associated with prevention or attenuationof intracellular membrane fusion. The fusion complex may comprise acellular hairpin membrane fusion protein. The cellular hairpin membranefusion protein may be a cellular SNARE protein. The multimeric coiledcoil bundle may be a 4 helix bundle. The intracellular membrane fusionmay be associated with secretion of a hormone, cytokine orneurotransmitter.

The invention also includes a method of treating, attenuating orpreventing HIV infection, the method comprising administering to apatient a drug compound which disrupts one or more intra-molecular orinter-molecular hydrogen bonds of the hydrogen-bonded ring structure ofgp41 trimer, wherein disruption of the hydrogen-bonded ring structure isassociated with attenuation or prevention of HIV infection.

The invention also encompasses a synthetic helical peptide wherein thepeptide sequence binds specifically to at least a fragment of the N-36helix of gp41, wherein the fragment includes the residue Q655 andwherein binding of the synthetic helical peptide to the N-36 helixdisrupts hydrogen bonded ring structure between the N36 and C34 helicesof gp41.

The invention also encompasses a peptidomimetic drug that bindsspecifically to helical sequences adjacent to the Q655 or Q553 residuesof gp41, and disrupts or prevents the formation of a hydrogen bondedring structure involving Q655 from the C34 helix and Q553 from the N36helix. The peptide or peptidomimetic binds to or interacts with the N36or C34 helices of gp41 and thereby disrupts one or more intra-molecularor inter-molecular hydrogen bonds of the hydrogen-bonded ring structureof gp41 trimer, wherein disruption of the hydrogen-bonded ring structureis associated with attenuation or prevention of HIV infection. Thepeptide or peptidomimetic may disrupt one or more of the intra-molecularor inter-molecular hydrogen bonds stabilizing the multimeric coiled coilbundle in the activated fusion complex required for the fusion andrelease of synaptic vesicles. It may also disrupt one or more of theintra-molecular or inter-molecular hydrogen bonds stabilizing themultimeric coiled coil bundle structurally homologous to the N36 or C34helix of HIV in the activated fusion complex required for the fusion andrelease of vesicles or granules containing pro-inflammatory proteins,cytokines, hormones, or vasoactive substances.

The invention also encompasses a method of attenuating or preventingHIV-1 infection comprising administering to a patient an effectiveamount of an agent which disrupts one or more intra-molecular orinter-molecular hydrogen bonds of the hydrogen-bonded ring structure ofgp41 trimer, wherein disruption of the hydrogen-bonded ring structuremakes HIV-1 susceptible to neutralization by the patient's antibodieswhich thereby attenuate or prevent HIV infection. The agent may comprisea peptide or peptidomimetic compound.

The invention also encompasses a peptide having a formula selected fromthe group consisting of:

(SEQ ID NO: 1) X-YTSLIHSLIEESQNQ[*]EKNEQELLELDKWASLWNWF-Z (SEQ ID NO: 2)X-YTNTIYTLLEESQNQ[*]EKNEQELLELDKWASLWNWF-Z (SEQ ID NO: 3)X-YTGIIYNLLEESQNQ[*]EKNEQELLELDKWANLWNWF-Z (SEQ ID NO: 4)X-YTSLIYSLLEKSQIQ[*]EKNEQELLELDKWASLWNWF-Z (SEQ ID NO: 5)X-LEANISKSLEQAQIQ[*]EKNMYELQKLNSWDIFGNWF-Z and (SEQ ID NO: 6)X-LEANISQSLEQAQIQ[*]EKNMYELQKLNSWDVFTNWL-Z

For the above listed peptides, amino acid residues are presented by thesingle-letter code; X comprises an amino group, an acetyl group, a9-fluorenylmethoxy-carbonyl group, a hydrophobic group, or amacromolecule carrier group; Z comprises a carboxyl group, or an amidogroup, or a hydrophobic group, or a macromolecular carrier group; and[*] represents any amino acid other than Q or N. In some embodiments [*]represents R, K, S, or E.

Another discovery of potential relevance to the understanding of thedeterminants of neutralization sensitivity and resistance of HIV-1 isdisclosed in J. Virol. doi:10.1128/JVI.00790-10, ‘Mutation at a singleposition in the V2 domain of the HIV-1 envelope protein confersneutralization sensitivity to a highly neutralization resistant virus’by Sara M. O'Rourke, Becky Schweighardt, Pham Phung, Dora P. A. J.Fonseca, Karianne Terry, Terri Wrin, Faruk Sinangil, and Phillip W.Berman, hereby incorporated by reference. In this work the authors madeuse of the swarm of closely related envelope protein variants(quasispecies) from an extremely neutralization resistant clinicalisolate in order to identify mutations that conferred neutralizationsensitivity to antibodies in serum from HIV-1 infected individuals. Theauthors describe a virus with a rare mutation at position 179 in the V2domain of gp120, where replacement of aspartic acid (D) by asparagine(N) converts a virus that is highly resistant to neutralization bymultiple polyclonal and monoclonal antibodies, as well as antiviralentry inhibitors, to one that is sensitive to neutralization. Althoughthe V2 domain sequence is highly variable, D at position 179 is highlyconserved in HIV-1 and SIV and is located within the LDI/V recognitionmotif of the recently described alpha-4B7 receptor binding site. Ourresults suggest that the D179N mutation induces a conformational changethat exposes epitopes in both the gp120 and gp41 portions of theenvelope protein such as the CD4 binding site and the MPER that arenormally concealed by conformational masking. These results suggest thatD179 plays a central role in maintaining the conformation andinfectivity of HIV-1 as well as mediating binding to alpha-4-beta-7({acute over (α)}4β7).

Additionally, the inventors have discovered (SC2009-117) certainprotease cleavage sites in HIV glycoproteins occur in regions importantfor receptor binding and the binding of neutralizing antibodies. Theinventors believe that HIV has developed a mechanism of immune escapeinvolving incorporation of protease cleavage sites in regions of themolecule important for the formation and binding of neutralizingantibodies. It is believed that these sites cause critical epitopes to“self destruct” before they can stimulate effective immune responses.The inventors disclose methods that use inhibition of proteolysis at oneor more of these cleavage sites to enhance the immunogenicity of HIVantigens. This is believed to be an entirely novel approach to treatingand preventing HIV infection.

For the sake of ease of reference during prosecution, the applicantherein sets out a number of defined inventions in claim-like format,taken from the priority applications. These are not claims, althoughthey have a claim format. The claims appear, as usual, at the end of theapplication.

Claims from SC2009-449

1. A method for screening drug candidates to identify a drug thatprevents or attenuates the ability of HIV gp41 to mediate cell fusionwith a CD4+ cell, the method comprising the following steps:

a) providing a putative drug candidate

b) providing a membrane-bound trimeric HIV gp41 trimer

c) measuring the integrity of the hydrogen-bonded ring structure of theHIV gp41 trimer

d) contacting the membrane-bound trimeric HIV gp41 with the putativedrug candidate

e) re-measuring the integrity of the hydrogen-bonded ring structure ofthe HIV gp41 trimer

f) whereby degree of integrity of the hydrogen-bonded ring structure ofthe HIV gp41 trimer is proportional to the ability of HIV gp41 tomediate cell fusion with a CD4+ cell, and wherein the ability of themembrane-bound trimeric HIV gp41 to mediate cell fusion is reduced bydisruption of the hydrogen-bonded ring structure of the HIV gp41 trimer.2. The method of claim 1 wherein the membrane-bound trimeric HIV gp41 isbound in the membrane of a virus, a pseudovirus, or a transfected cell.3. The method of claim 1 wherein disruption of the hydrogen-bonded ringstructure results in exposure of previously hidden epitopes which bindspecifically with broadly neutralizing antibodies.4. The method of claim 1 wherein the drug candidate is an antibody thatbinds specifically with an epitope of gp41 or a neutralizing antibodythat targets gp120.5. A method for increasing the immunogenicity of HIV envelope proteinsthe method comprising exposing the HIV virus to a drug compound thatdisrupts the hydrogen bonded ring structure between the N36 and C34helices of gp41.6. The method of claim 1 of 5 wherein the drug compound is a smallmolecule.7. The method of claim 1 of 5 wherein the drug compound is an antibody.8. The method of claim 1 of 5 wherein the drug compound is an inhibitorof CD4 binding selected from the group consisting of: 4E10, 2F5, Q665R,Q655K and Q655E.9. The method of claim 1 or 5 wherein the drug compound is an inhibitorof HIV fusion binding and becomes a more effective inhibitor in thepresence of a molecule that disrupts the disulfide bonded ring structureof gp41.10. The method of claims 1 or 5 wherein the drug compound disrupts thehydrogen bonded ring structure between the N36 and C34 helices of gp41and thereby exposes neutralizing epitopes which are recognized byendogenous or exogenous antibodies which then are able to neutralize thevirus.11. A method for screening for a drug that prevents or attenuatesintracellular membrane fusion, the method comprising exposing themultimeric coiled coil bundle of the activated fusion complex to a drugcandidate, wherein the fusion complex comprises a cellular hairpinmembrane fusion protein, wherein disruption of one or more hydrogenbonds of the fusion complex is associated with prevention or attenuationof intracellular membrane fusion.12. The method of claim 11 wherein the intracellular membrane fusion isassociated with secretion of a hormone, cytokine or neurotransmitter.13. A method of treating, attenuating or preventing HIV infection, themethod comprising administering to a patient a drug compound whichdisrupts one or more intra-molecular or inter-molecular hydrogen bondsof the hydrogen-bonded ring structure of gp41 trimer, wherein disruptionof the hydrogen-bonded ring structure is associated with attenuation orprevention of HIV infection.14. A synthetic helical peptide wherein the peptide sequence bindsspecifically to at least a fragment of the N-36 helix of gp41, whereinthe fragment includes the residue Q655 and wherein binding of thesynthetic helical peptide to the N-36 helix disrupts hydrogen bondedring structure between the N36 and C34 helices of gp41.15. A peptidomimetic drug that binds specifically to helical sequencesadjacent to the Q655 or Q553 residues of gp41, and disrupts of preventsthe formation of a hydrogen bonded ring structure involving Q655 fromthe C34 helix and Q553 from the N36 helix.16. The peptide or peptidomimetic compound of claim 14 wherein thecompound binds to or interacts with the N36 or C34 helices of gp41 andthereby disrupts one or more intra-molecular or inter-molecular hydrogenbonds of the hydrogen-bonded ring structure of gp41 trimer, whereindisruption of the hydrogen-bonded ring structure is associated withattenuation or prevention of HIV infection.17. The peptide or peptidomimetic compound of claim 14 wherein thecompound disrupts one or more of the intra-molecular or inter-molecularhydrogen bonds stabilizing the multimeric coiled coil bundle in theactivated fusion complex required for the fusion and release of synapticvesicles.18. The peptide or peptidomimetic compound of claim 14 wherein thecompound disrupts one or more of the intra-molecular or inter-molecularhydrogen bonds stabilizing the multimeric coiled coil bundlestructurally homologous to the N36 or C34 helix of HIV in the activatedfusion complex required for the fusion and release of vesicles orgranules containing pro-inflammatory proteins, cytokines, hormones, orvasoactive substances.19. A method of attenuating or preventing HIV-1 infection comprisingadministering to a patient an effective amount of an agent whichdisrupts one or more intra-molecular or inter-molecular hydrogen bondsof the hydrogen-bonded ring structure of gp41 trimer, wherein disruptionof the hydrogen-bonded ring structure makes HIV-1 susceptible toneutralization by the patient's antibodies which thereby attenuate orprevent HIV infection.20. The method of claim 19 wherein the agent comprises a peptide orpeptidomimetic compound.21. A peptide having a formula selected from the group consisting of:

(SEQ ID NO: 1) X-YTSLIHSLIEESQNQ[*]EKNEQELLELDKWASLWNWF-Z (SEQ ID NO: 2)X-YTNTIYTLLEESQNQ[*]EKNEQELLELDKWASLWNWF-Z (SEQ ID NO: 3)X-YTGIIYNLLEESQNQ[*]EKNEQELLELDKWANLWNWF-Z (SEQ ID NO: 4)X-YTSLIYSLLEKSQIQ[*]EKNEQELLELDKWASLWNWF-Z (SEQ ID NO: 5)X-LEANISKSLEQAQIQ[*]EKNMYELQKLNSWDIFGNWF-Z, and (SEQ ID NO: 6)X-LEANISQSLEQAQIQ[*]EKNMYELQICLNSWDVFTNWL-Z,in which:amino acid residues are presented by the single-letter code;X comprises an amino group, an acetyl group, a9-fluorenylmethoxy-carbonyl group, a hydrophobic group, or amacromolecule carrier group;Z comprises a carboxyl group, an amido group, a hydrophobic group, or amacromolecular carrier group. [*] represents any amino acid other than Qor N.22. The peptide of claim 21 wherein [*] represents R, K, S, or E.Claims from UCSC 2010-1171. A vaccine formulation comprising an HIV envelope glycoprotein and aprotease inhibitor.2. The vaccine formulation of claim 1 wherein the protease inhibitor isa cathepsin.3. The vaccine formulation of claim 2 wherein the cathepsin is humancathepsin L, S or D.4. The vaccine formulation of claim 1 formulated with an excipient,carrier or adjuvant for use as a vaccine.5. A vaccine formulation comprising an HIV envelope glycoprotein whereinone or more conserved cleavage sites of the HIV envelope glycoprotein isprotected from protease cleavage by in vitro mutagenesis, and whereinthe one or more conserved cleavage sites is selected from the cathepsincleavage sites of MN-rgp120 and variants thereof.6. The vaccine formulation of claim 5 wherein said vitro mutagenesisresults in deletion, mutation, methylation or acetylation at a conservedcleavage site.7. The vaccine formulation of claim 5 wherein the cathepsin cleavagesites are selected from the group consisting of SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14 and SEQ ID NO:15.8. A method for treatment or prevention of a viral infection, the methodcomprising administering to a subject the vaccine formulation comprisingan HIV envelope glycoprotein wherein one or more conserved cleavagesites of the HIV envelope glycoprotein is protected from proteasecleavage by in vitro mutagenesis resulting in deletion, mutation,methylation or acetylation, and wherein the one or more conservedcleavage sites is selected from the cathepsin cleavage sites ofMN-rgp120 and variants thereof.9. The method of claim 8 wherein the cathepsin cleavage sites areselected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 and SEQ ID NO:15.10. The method of claim 8 further comprising contemporaneouslyadministering to a subject a protease inhibitor.11. The method of claim 10 wherein the protease inhibitor is aninhibitor of a cathepsin.12. The method of claim 11 wherein the protease inhibitor inhibitscleavage at the cathepsin cleavage sites of MN-rgp120.13. A method for provoking an immune response in a mammal, the methodcomprising administering to said mammal an HIV envelope glycoproteinwherein one or more conserved cleavage sites of the HIV envelopeglycoprotein is protected from protease cleavage by in vitromutagenesis, and wherein the one or more conserved cleavage sites isselected from the cathepsin cleavage sites of MN-rgp120 as shown inTable 1, and variants and/or homologues thereof.14. An isolated HIV polynucleotide encoding a protected virus envelopeprotein wherein one or more conserved cleavage sites of the HIV envelopeglycoprotein is protected from protease cleavage by in vitromutagenesis, and wherein the one or more conserved cleavage sites isselected from the cathepsin cleavage sites of MN-rgp120 and variantsthereof15. The isolated polynucleotide of claim 14 wherein the conservedprotease cleavage sites serve to inactivate epitopes recognized byneutralizing antibodies and are responsible for the lack of protectiveimmune responses when used as a vaccine antigen.16. The isolated polynucleotide of claim 14 wherein conserved cleavagesites that are recognized by serum or cellular proteases are deleted orinactivated by in vitro mutagenesis.17. The isolated polynucleotide of claim 14 wherein in vitro mutagenesisof conserved cleavage sites protects the neutralizing epitopes fromproteolytic degradation after parenteral injection.18. The isolated polynucleotide of claim 14 wherein conserved proteasecleavage sites located within epitopes recognized by neutralizingantibodies are deleted or inactivated by in vitro mutagenesis in such away as to preserve the ability to bind neutralizing antibodies.19. The isolated polynucleotide of claim 14 wherein the proteasecleavage sites are specific for the antigen processing enzymes:cathepsin L, cathepsin S, or cathepsin D.20. The isolated polynucleotide of claim 14 wherein the proteasecleavage sites are specific for the serum protease thrombin, or the cellassociated protease, tryptase, or the inflammation associated proteasessuch as elastase.21. The isolated polynucleotide of claim 14 wherein the proteasecleavage sites are specific for cathepsin B, K, or N.22. The isolated polynucleotide of claim 14 wherein the protein consistsof monomeric or oligomeric fragments of the HIV envelope protein gp160such as gp120, gp140, or gp41.23. The isolated polynucleotide of claim 14 wherein the protein consistsof monomeric or oligomeric fragments of the influenza virushaemagglutinin (HA1/HA2) of any strain of influenza (e.g. H1N1).24. The isolated polynucleotide of claim 14 wherein the protein consistsof monomeric or oligomeric fragments of glycoprotein D from HerpesSimplex virus type 1 or 2.25. The isolated polynucleotide of claim 14 wherein the protein consistsof monomeric or oligomeric fragments of any virus envelope protein forcellular receptor binding.Claims from UCSC2008-7761. A method of analyzing intra-patient HIV virus variation to identifyspecific amino acid residues of the HIV envelope glycoproteins thataffect sensitivity or resistance to broadly neutralizing antibodies, themethod comprising the steps of:i) providing a plurality of individual subjects who are seropositive forHIV antibodies and taking a biological sample from each subject, whereinthe sample contains a multiplicity of HIV viruses with closely relatedgenomes, wherein all subjects had been infected with HIV no more thanone year before, and no less than one month before sample collection,ii) amplifying the env genes of the multiplicity of viruses to produce alibrary of different env genes,iii) cloning the amplified env genes into a plasmid shuttle vector thatallows the plasmid to replicate in both bacteria and mammalian cells,iv) transforming bacterial cells with the shuttle vector and plating outthe transformed bacterial cells onto a selective medium so that bacteriacontaining the shuttle vector plasmid containing the cloned envelopegene are selectable,v) selecting individual colonies at random and preparing plasmid DNAfrom each colony selected and analyzing the plasmid DNA by restrictiondigestion so as to identify plasmids containing the full length HIVenvelope gene, which plasmids are used to produce pseudoviruses,vi) co-transfecting mammalian cells with the env-containing vector andsimultaneously with a plasmid containing a defective HIV provirusplasmid where the coding sequence of the env gene has been replaced withthe coding sequence of a marker gene, and culturing the co-transfectedmammalian cells in a culture medium, to produce pseudovirions containingthe amplified env genes, which pseudovirions are released into the cellculture medium,vii) harvesting the supernatant from the cell culture medium, whereinthe supernatant contains pseudoviruses from the transfected cells, andwherein each supernatant contains a stock of pseudovirus resulting froma single purified expression plasmid,viii) testing the pseudovirion from the selected colonies to determineinfectivity by culturing the pseudovirions with cells capable of beinginfected by HIV, wherein infectivity is measured by the degree ofexpression of the marker gene,ix) selecting pseudovirions that exhibit high infectivity, and testingthe selected pseudovirions for sensitivity or resistance toneutralization by one or more broadly neutralizing antibodies,x) selecting pairs of plasmids from the same individual wherein eachpair contains at least one neutralization resistant and at least oneneutralization sensitive pseudovirus,xi) sequencing the envelope genes identified from sensitive andresistant pseudovirus pairs,xii) comparing the nucleotide sequences of the envelope genes of theneutralization sensitive and resistant pairs thereby identifyingspecific amino acid differences between the pairs and identifyingpolymorphisms that may affect sensitivity or resistance toneutralization by broadly neutralizing antibodies,xiii) at each amino acid residue that differs between the neutralizationsensitive and neutralization resistant envelope genes, site-by-sitereplacement of amino acids from the is performed, substituting one aminoacid at a time from neutralization sensitive sequence into theneutralization resistant sequence,xiv) each new construct is used to create a pseudotype virus which istested for neutralization sensitivity so as to identify specific aminoacid residues of the HIV envelope glycoproteins that affect sensitivityor resistance to broadly neutralizing antibodies.2. The method of claim 1 wherein all subjects had been infected with HIV109 days+/−58 days before specimen collection.3. A vaccine composition comprising an HIV envelope glycoprotein whereina glutamine residue at a site identifiable as being homologous toposition 655 of SEQ ID NO:16 is replaced by a substitute amino acid suchthat the amino acid substitution disrupts an inter-molecularhydrogen-bonded ring structure between the N36 and C34 helices of thegp41 trimer.4. The vaccine composition of claim 3 wherein possession of the HIVenvelope glycoprotein confers greater neutralization sensitivity upon anHIV virus when it is exposed to 2F5 or 4E10 monoclonal antibodies,Enfuvirtide or CD4-IgG, than would be provided by another HIV envelopeglycoprotein identical in all respects except for the substitution ofthe glutamine residue.5. The vaccine composition of claim 3 or 4 wherein the substitute aminoacid is arginine.6. The vaccine composition of claim 3 or 4 wherein the substitute aminoacid is Lysine, Serine or Glutamic acid.7. The vaccine composition of claims 3, 4, 5 or 6 wherein the HIVenvelope glycoprotein has at least 60% sequence identity to SEQ IDNO:16.8. The composition of claims 3, 4, 5 or 6 wherein the HIV envelopeglycoprotein comprises a fusion protein that includes a non-HIV signalsequence and a flag epitope.9. The vaccine composition of claims 3, 4, 5 or 6 wherein the HIVenvelope glycoprotein has had a furin cleavage site deleted.10. The vaccine composition of claim 3 or 4 wherein the HIV envelopeglycoprotein comprises a full length gp160 wherein a glutamine residueat a site identifiable as being homologous to position 655 of SEQ IDNO:16 is replaced by arginine.11. The vaccine composition of claims 3, 4, 5 or 6 wherein thepolypeptide comprises a truncated form of the envelope protein lackingthe gp41 transmembrane domain and cytoplasmic tail.12. A polynucleotide encoding an HIV envelope glycoprotein wherein aglutamine residue at a site identifiable as being homologous to position655 of SEQ ID NO:16 is replaced by a substitute amino acid such that theamino acid substitution disrupts an inter-molecular ring structurebetween the N36 and C34 helices of the gp41 trimer.13. The polynucleotide of claim 14 formulated in an vector as a DNAvaccine.14. A method for inhibiting the fusion of an HIV virus to a host cell,the method comprising exposing the HIV virus to a compound that disruptsthe hydrogen-bonded ring structure between the N36 and C34 helices ofgp41.15. A method for increasing the immunogenicity of HIV envelope proteinsthe method comprising exposing the HIV virus to a compound that disruptsthe hydrogen bonded ring structure between the N36 and C34 helices ofgp41.16. The method of claim 14 or 15 wherein the compound is a smallmolecule.17. The method of claim 14 or 15 wherein the compound is an antibody.Brief Description of the Figures (SC2009-449)

FIG. 1. Mutation of neutralization resistant clone 022 from subject108060 (A) Amino acids from neutralization-resistant clone 022 are shownas open rectangles. Amino acids from neutralization-sensitive clone 024were inserted by in vitro mutagenesis, and are shown as shadedrectangles. (B) Schematic showing the position of the Q655R mutation inrelation to the entry inhibitor Fuseon (or T-20 peptide), the MPER, andpeptides recognized by the broadly neutralizing monoclonal antibodies2F5 and 4E10. The locations of gp41 structural elements are shown asfollows: shaded boxes for the hydrophobic fusion domain (FD), thetransmembrane domain (TMD), and the MPER. Sequences defining the C34 andN36 helices are shown as open rectangles (Sequences are as follows: C34peptide, SEQ ID NO:36; Fuseon (T-20), SEQ ID NO:37; and MPER, SEQ IDNO:38).

FIG. 2. Conformational transitions involved formation of the fusionactive 6 helix bundle in gp41. Binding of the HIV envelope protein gp120by CD4 and either of the CXCR4 or CCR5 chemokine receptors (agonists)triggers the formation of the pre-hairpin intermediate structure. Thistransition is inhibited by antagonists such as CD4 blocking antibodiesfound in HIV+ sera and the CD4 blocking MAb, b12. The transition fromthe pre-hairpin intermediate to the fusion active 6 helix bundlestructure is facilitated by cooperative interactions between the N-36and C34 helices and the hydrogen bonded ring structure involving Q655.This transition is antagonized by bNAbs in HIV+ sera, MAbs such as 2F5and 4E10, the antiviral drug, FUZEON, and mutations such as Q655R,Q655K, Q655S, and Q655E that destabilize the hydrogen bonded ringstructure of the 6 helix bundle.

FIG. 3. shows the method of swarm analysis. Swarm analysis ofquasi-species from one individual infected with HIV, for theidentification of mutations that confer sensitivity and resistance tobroadly neutralizing antibodies.

FIG. 4. shows the results of the infectivity screen used to identify envclones used in the neutralization assay. Infectivity screen to identifyenvelope clones for use in neutralization assay. 24-48 envelope genesare isolated from each subject and used to construct pseudoviruses.Viruses are screened for infectivity on CD4⁺ cell lines expressingeither the CCR5 or CXCR4 chemokine receptors to determine the tropism ofeach envelope clone.

FIG. 5. shows the location of amino acid differences between sensitiveand resistant clones.

FIG. 6. shows graphs of neutralization for various clones.Neutralization of mutants from clone 022 of subject 108060. The mutantenvelope genes described in FIG. 1 were used to construct pseudoviruseswhich were evaluated for sensitivity to neutralization by HIV+ serum.Curves for the Z23 serum are shown. The ordinate indicates percentageneutralization and the abscissa indicates the log of the serum dilution.The dotted line indicates the 50% neutralization titer. For brevity, thecytoplasmic tail mutants are not shown, but all had well behavedneutralization curves.

Brief Description of the Figures (SC 2010-117)

FIG. 7. Cathepsin digestion of MN-rgp120. MN-rgp120 was digested witheither cathepsin L (panel A), cathepsin S (panel B), or cathepsin D(panel C). At the indicated times, samples were removed, the digestionwas stopped by the addition of liquid nitrogen, and prepared forSDS-PAGE analysis. Bands were visualized by Coomassie blue staining. Themobilities of the fragments identified are shown in the outside lanes.

FIG. 8. Diagram of MN-rgp120 fragments generated by cathepsin digestion.MN10-rgp120 was digested with cathepsin L, S, or D and the resultingfragments were purified and analyzed by Edmund sequence degradation.Solid lines indicate peptides that were resolved by polyacrylmide gelelectrophoresis and characterized by N-terminal sequence analysis.Dashed lines indicate the location of peptides deduced from mobility andsequence analysis, but not recovered.

FIG. 9. Location of cathepsin L, S, and D cleavage sites on MN-rgp120disulfide bonded schematic. Cathepsin L, S and D cleavage sites inMN-rgp120 were identified by Edman sequence degradation and located ontothe disulfide bonded structure of gp120 determined by Leonard et al.1990 (50). Cathepsin L sites are indicated by a closed arrow, cathepsinS sites are indicated by a line arrow, and cathepsin D sites areindicated by an open arrow. The location of amino acid residues reportedto be important for the binding of CD4, chemokine receptors, and the α4βintegrin are indicated by residues shaded red, dark blue, and lightblue, respectively. The location of amino acids known to be importantfor the binding of the broadly neutralizing antibody, b12, andneutralizing antibodies to the V3 domain are indicated by amino acidsshaded green and purple, respectively. Residues with two or more colorsindicate amino acids involved in the binding of two or more receptors ofneutralizing monoclonal antibodies. The figure was created based onresults from Kwong et al.; Zhou et al., Rizzuto et al.; Decker et al.and Arthos et al. (2). The numbering provided is based on the sequenceof gp120 from the MNGNE isolate of HIV (SEQ ID NO:22).

FIG. 10. Location of cathepsin L, S and D cleavage sites on 3-Dstructure of gp120 bound to CD4. The locations of cathepsin cleavagesites were located on the 3-dimensional structure of gp120 based on thestructure of a gp120 fragment complexed with CD4 described by Huang etal. 2005. Cathepsin L sites are indicated in green; cathepsin S sites inred; and cathepsin D sites in blue. The structure of CD4 is shown inyellow. Numbering is based on the sequence of MN-rgp120.

FIG. 11. Antibody binding to cathepsin L and D treated gp120. PurifiedMN17 rgp120 was treated with either cathepsin L or cathepsin D andcaptured onto the sur18 face of microtiter plates coated with apolyclonal antibody, D7324, directed to the C ter19 minus of gp120.Monoclonal antibodies were incubated with the gp120 coated microtiterplates and binding was determined by ELISA. Closed symbols indicate thebinding to untreated gp120; open circles represent binding tocathepsin-L treated gp120; open squares indicate binding to cathepsin-Dtreated gp120.

FIG. 12. (from SC-2008-776): Gp41 Functional Domains and Comparison ofSequences of Functionally Significant Regions of the N36 and C34 Helices(Sequences are as follows: N36 (546-581), SEQ ID NO:41; C34 (628-661),SEQ ID NO:36; T-20 (638-673), SEQ ID NO:1; and CHR-5 (643-678), SEQ IDNO:42).

FIG. 13. Alignment of predicted and observed cathepsin L, S and Dcleavage sites. Reference sequences for gp120 were obtained from theLosa Alamos HIV database. Env sequences from clades A, C, D and E (crfA/E) as well as reference sequences for chimpanzee isolates of HIV andSIV were aligned with the sequences of prototypic clade B MN and HXB2strains of HIV using the MAFFT sequence alignment program. Numbering isprovided with reference to the MN strain. Sequences shown begin with themature amino terminus of gp120. Predicted and observed cathepsin Lcleavage sites are indicated by open bars and closed bars respectively.Cathepsin L cleavage sites are indicated in green, cathepsin S sites inred, and cathepsin D sites in blue. The full names of the alignedsequences are: MN (MN-rgp120, the sequence corresponds to residue 26 to507 of SEQ ID NO:22); HXB2 (the sequence corresponds to residue 27 to507 of SEQ ID NO:23); A1.KE (A1.KE.94.Q23_17, the sequence correspondsto residue 26 to 492 of SEQ ID NO:24); A1.UG (A1.UG.92.92UG037, thesequence corresponds to residue 26 to 496 of SEQ ID NO:25); C.ET(CET.86.ETH2220, the sequence corresponds to residue 26 to 491 of SEQ IDNO:26); C.IN (C.IN.93.93IN101, the sequence corresponds to residue 26 to503 of SEQ ID NO:27); D.TZ (D.TZ.01.A208, the sequence corresponds toresidue 26 to 498 of SEQ ID NO:28); D.UG (D.UG.94.94UG114, the sequencecorresponds to residue 26 to 490 of SEQ ID NO:29); AE.T93(AE.TH.93.93.TH051, the sequence corresponds to residue 26 to 499 of SEQID NO:30); AE.T90 (AE.TH.90.CM240, the sequence corresponds to residue26 to 496 of SEQ ID NO:31); CPZ.05 (CPZ.CM.05.SIVcpzMT145, the sequencecorresponds to residue 26 to 485 of SEQ ID NO:32): CPZ85(CPZ.US.85.CPZUS, the sequence corresponds to residue 21 to 473 of SEQID NO:33); SVV51 (SIV.US.MAC251, the sequence corresponds to residue 17to 518 of SEQ ID NO:34); SIV39.US.MAC239, the sequence corresponds toresidue 17 to 516 of SEQ ID NO:35).

FIG. 14 shows three pairs of sequences from neutralization sensitive andneutralization resistant viruses. The sequences shown are envelopesequences from: subject 108060 (Panel A: p1.10848_c2 Resistant, SEQ IDNO: 43; Panel B: p1.10848_c11 Sensitive, SEQ ID NO: 44); subject 108051(Panel C: 108051_c6 Sensitive, SEQ ID NO: 45; Panel D: p1. 108051_c5Resistant, SEQ ID NO: 46); and subject 108060 (Panel E: p1. 108060_c22Resistant, SEQ ID NO: 47; Panel F: p1. 108060_c24 Sensitive, SEQ IDNO:48).

GENERAL REPRESENTATIONS CONCERNING THE DISCLOSURE

This specification incorporates by reference all documents referred toherein and all documents filed concurrently with this specification orfiled previously in connection with this application, including but notlimited to such documents which are open to public inspection with thisspecification.

Definitions

The terms “amino acid” and “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these.

“Amplification” relates to the production of additional copies of anucleic acid sequence e.g., using polymerase chain reaction (PCR).

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant.

The term “similarity” refers to a degree of complementarily. There maybe partial similarity or complete similarity. The word “identity” maysubstitute for the word “similarity.” A partially complementary sequencethat at least partially inhibits an identical sequence from hybridizingto a target nucleic acid is referred to as “substantially similar.”

The phrase “percent identity” as applied to polynucleotide orpolypeptide sequences refers to the percentage of residue matchesbetween at least two sequences aligned using a standardized algorithmsuch as any of the BLAST suite of programs (e.g., blast, blastp, blastx,nucleotide blast and protein blast) using, for example, defaultparameters. BLAST tools are very commonly used and are available on theNCBI web site.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” tool setat default parameters. Such a pair of polypeptides may show, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 86%, at least 90%, at least 95%, or at least 98% or greatersequence identity over a certain defined length of one of thepolypeptides.

Detailed Description of the Invention (SC2009-449 PCT)

Described is a new way to identify mutations that effect sensitivity andresistance to virus neutralization by anti-HIV antisera. Some of thesemutations occur in previously undescribed sites critical forpreservation of the structure and function of HIV. One of these sitesappears to affect a previously overlooked hydrogen bonded ring structurein the trimeric form of the HIV envelope protein, gp41. This novelstructure is formed by oligomeric interactions between the C34 and N-36helices of gp41 and is located close to the C-terminus of the domainsthat undergo massive rearrangement to form the 6 helix bundle requiredfor virus entry and fusion. Disruption of this structure by naturallyoccurring or experimental mutations renders the virus much moresensitive to neutralization by antibodies. Disclosed is the developmentof and the use of small molecule drugs that target this site whichinterfere with virus fusion in such a way as to prevent, or lower theefficiency of fusion and therefore virus infection.

A mutation mapped and herein disclosed is located in the middle of asequence that forms the basis of a commercially marketed HIV antiviraldrug, FUZEON. The structure identified allows for the rational design ofnew compounds targeting the same area as FUZEON, but which work by adifferent mechanism.

The molecular structures responsible for HIV fusion have been conservedthrough evolution, and homologous structures are present in otherviruses, such as influenza, and vesicle proteins required for the exportand secretion of a number of important molecules (e.g. hormones,cytokines, an neurotransmitters). Targeting weak, hydrogen bondedinteractions of the type that we have identified may provide a newapproach to the development of small molecule therapeutics that disruptsuch structures.

Disclosed is a new method (“swarm analysis”) used to identify mutationsthat confer sensitivity and resistance to neutralization by bNAbs(broadly neutralizing antibodies) in polyclonal HIV+ sera with broadneutralizing activity. The method takes advantage of the swarm ofclosely related virus variants that occur in each HIV-infectedindividual to establish panels of envelope proteins that differ fromeach other by a limited number of mutations causing amino acidsubstitutions (1-3%). By studying the effect of these mutations inswarms of viruses from the same individual, we can identify specificamino acids that affect sensitivity and resistance to neutralization byHIV+ sera. We have used this method to identify a novel structuralelement in the gp41 fragment of the HIV envelope glycoprotein thatappears to stabilize the oligomeric 6 helix bundle in the HIV-1 fusionapparatus. This oligomeric 6 helix structure is important in promotingfusion of the viral membrane to membrane of the host cell beinginfected. Mutations that affect this structure confer sensitivity orresistance to virus neutralization, i.e., they make the virus more orless sensitive to neutralizing Abs such as broadly neutralizingantibodies.

The studies described made use of a large collection of clinicalspecimens from new and recent HIV infection collected in the course of aPhase 3 clinical trial (VAX004) of a candidate HIV-1 vaccine, AIDSVAXB/B (Flynn N M, Forthal D N, Harro C D, Judson F N, Mayer K H, Para M F;“Placebo-controlled phase 3 trial of a recombinant glycoprotein 120vaccine to prevent HIV-1 infection.” The Journal of infectious diseases2005; 191:654-65). This collection of specimens is unique in that theywere obtained within six months of infection and are representative ofviruses currently circulating in North America. Transmission of HIV-1involves a genetic bottleneck where, out of the myriad of geneticvariants in each HIV infected donor, only a single homogeneous variantof HIV-1 successfully replicates in the recipient. This variantreplicates to very high titers in the first days and weeks after HIV-1infection and eventually starts to mutate in response to error-pronereverse transcription to generate a swarm of closely related variants(Richman et al., 2003; Wei et al., 2003). The swarm of viruses furtherdiversifies in response to selective pressures imposed by both cellularand humoral antiviral immune responses and in response to drug therapy.Virus variation, driven by the relentless error-prone reversetranscription and selection by the immune system, occurs throughout thecourse of HIV infection, and is perhaps the greatest challenge in thedevelopment of vaccine and therapeutic products. The applicants reasonedthat by studying viruses from early infections, sequence variation wouldbe limited compared to sequences collected at later times. The analysisdescribed is made possible by high throughput, automated methods forvirus infectivity and neutralization assays as well as systems for theconstruction and analysis of pseudotype viruses (Schweighardt et al.,2007, J Acquir Immune Defic Syndr 46:1-11 and Whitcomb et al., 2007,Antimicrob Agents Chemother 51:566-75) with defined amino acidsequences. This technology allows for the accurate and efficientanalysis of thousands of individual envelope glycoproteins forsensitivity/resistance to neutralization by panels of HIV+ sera. Theseanalyses provide particular insight into the strategies employed by HIVto evade the immune response and can guide the development of a newgeneration of HIV vaccine antigens, one or more of which are describedherein.

Experimental Methods and Results

Cryopreserved plasma was obtained from 28 randomly selected individualswho became infected with HIV during the course of the VAX004 clinicaltrial. The specimens were all collected from the first postdiagnosisblood draw, with a mean estimated time post infection of 109+/−58 days.Populations of gp160 genes were amplified from each patient plasma byRT-PCR and ligated into a plasmid expression vector to create librariesof envelope genes (Schweighardt et al., 2007). A diagram that describesthe swarm analysis strategy is provided in FIG. 3. The plasmid librariesfrom each individual were then used to create pseudoviruses forneutralization assays. Because HIV infection is known to result in ahigh frequency of defective envelope genes, it was necessary to screenindividual clones for infectivity prior to performing virusneutralization assays. For this purpose 24-48 individual colonies wereselected from each library, and the plasmids from each used to constructpseudotype viruses for initial screening in infectivity and receptortropism assays. Data from these infectivity studies on a cell line(CCR5/CD4/U89) expressing CD4 and CCR5 are provided in the supplementalinformation (FIG. 4). Based on the results of this assay, sets of 10pseudotype viruses with robust infectivity were selected from eachindividual for use in a pseudotype virus neutralization assay. These 280pseudotype viruses were then tested for sensitivity/resistance toneutralization by a panel of four standard HIV+ sera (Z23, Z1679, Z1684,and N16) known from previous studies to possess bNAbs. The results ofthese studies provided insights into both virus variation and variationin the specificity of bNAbs in different HIV+ sera. Overall threedifferent neutralization phenotypes were observed in the viruses. Wefound that one individual (1/28) possessed viruses that were extremelyresistant to neutralization, such that none of the 10 clones weresensitive to neutralization by any of the HIV+ sera. Conversely we foundthat some individuals (3/28) possessed viruses that were extremelysensitive to neutralization, such that almost all of the clones weresensitive to neutralization by all four HIV+ sera. However, in themajority of the individuals (24/28), we found a mixture ofneutralization sensitive and resistant clones.

When the activities of the four HIV+ sera were compared, differences inthe apparent potency and specificity of the bNAbs were observed. Forexample in some cases (e.g. 108059) only one of the four sera was ableto neutralize the clones from a particular individual (Table 1A). Thisresult suggested that serum Z23 possessed at least one population ofneutralizing antibodies that was missing or under-represented in theantibodies from the other HIV+ sera. One particularly interestingpattern of neutralization was found in subject 108060 (Table 1B) whereall four HIV+ sera neutralized three of the ten clones. These resultsraised the possibility of a mutational difference between clones thataffected a population of neutralizing antibodies common to all four HIV+sera. Because we expected sequence variation between clones from thesame individual to be minimal, we reasoned that comparison of thesequences between the neutralization sensitive and resistant variantswould allow us to identify the mutation that conferred neutralizationsensitivity.

Further examination of the dataset revealed that 7/28 individualsexhibited a similar pattern of neutralization sensitivity, where atleast one clone was sensitive to neutralization by all four HIV+ seraand at least one clone was resistant to all four HIV+ sera. Based onthis observation, we selected pairs of viruses (one neutralizationsensitive, and the other neutralization resistant) from seven of the 28individuals with the largest differences in neutralization titers forfurther analysis.

We next sequenced the envelope glycoproteins from each neutralizationsensitive/resistant pair and compared the sequences. In some cases wefound that sequence variation was minimal between the two clones fromthe same individual, whereas in other cases there were a large number ofamino acid differences between neutralization sensitive and resistantclones (FIG. 5). In one case (subject 108048), there were only two aminoacid differences between the neutralization sensitive and resistantclones. In contrast, other viruses (e.g. 108068) showed a large numberof amino acid differences (48) between neutralization sensitive andresistant viruses. Pairs with limited sequence variation allowed for thepossibility of in vitro mutagenesis to localize the amino acidsresponsible for conferring sensitivity or resistance to neutralizationby HIV+ sera. To explore this possibility, we initially selected theviruses from subject 108060 for further analysis.

Identification of a mutation in gp160 from subject 108060 that conferssensitivity to neutralization by HIV+ sera. It can be seen (Table 1A)that three of the ten clones from subject 108060 (clones 002, 018, and024) were sensitive to neutralization by all four HIV+ sera, and of theremaining seven clones, most were resistant to neutralization by HIV+sera Z1679, Z1684, and N16, but somewhat sensitive to HIV+ sera fromZ23. Based on the fact that there was at least a 10-fold difference inneutralization sensitivity with all four HIV+ sera, clones 022 and 024were selected for further study. When the gp160 sequences of theneutralization resistant variant (clone 022 wtR) and a neutralizationsensitive variant (clone 024 wtS) were compared (FIG. 5), it was foundthat they differed at only seven positions. Two of the amino aciddifferences were in gp120, two amino acid differences were in the gp41ectodomain, and the remaining three differences were in the cytoplasmictail of gp41. To determine which amino acids were responsible for thedifference in sensitivity to neutralization between clone 022 and clone024, a series of mutant envelope proteins were constructed and used tocreate pseudovirions where polymorphisms from the neutralizationsensitive variant (clone 024) were introduced into the neutralizationresistant (clone 022) background (FIG. 1A).

We found (Table 2A) that the replacement of asparagine (N) for serine(S) at position 323 (N323S) in the V3 domain of gp120 had no effect onsensitivity to neutralization. Similarly, the substitution of N forglycine (G) at position 530 in the C5 domain (N530G) of gp120 had noeffect. Replacement of lysine (K) at position 634 of the second heptadrepeat domain (C34 helix) of gp41 with glutamic acid (E) in the mutantK634E also failed to show a significant difference in neutralizationsensitivity.

However, the replacement of glutamine (Q) for arginine (R) at position655 (Q655R) resulted in a remarkable increase (>30 fold) inneutralization sensitivity by all four of the HIV+ sera.

Mutations in the cytoplasmic tail region (832/833 and 827/832/833) werealso examined and had no significant effect. The primary data used tocalculate 50% neutralization titers with HIV+ serum Z23 are presented insupplemental information (FIG. 6). It can be seen that theneutralization curves were well behaved for all of the mutants.

Localization of residue 655 on linear sequence and 3-D structure ofgp41. To better understand the impact of this mutation on the structureand function of the 108060 envelope glycoprotein, we located residue 655on the linear sequence and 3-dimensional structure of gp41. Examinationof the linear sequence (FIG. 1B) revealed that position 655 was locatedin the conserved second heptad repeat (HR2) of gp41 in a region alsoknown as the C34 helix. This part of the molecule is known to play anintegral role in virus fusion and indeed forms an essential component ofthe 6 helix bundle in the trimeric structure of gp41 that mediatesfusion of the viral membranes with cellular membranes in the course ofHIV infection. Position 655 is also located within in the T20 peptidethat serves as the basis for the antiviral drug, FUZEON, that inhibitsHIV infectivity by inhibiting virus fusion and entry. Finally thelocation of this mutation is only eight amino acids from a distinctstructural region of gp41, termed the Membrane Proximal External Region(MPER), that is known to contain distinct epitopes recognized by thebroadly neutralizing monoclonal antibodies 2F5, 4E10 and Z13. Takentogether, these results suggest that this mutation occurs in a regionthat is important for virus fusion, and is close to—but structurallydistinct from—a region known to contain other epitopes recognized bybNAbs. Interestingly, while the Q655R mutation in the C34 helix of gp41had a marked effect on virus neutralization, the K634E mutation also inthe C34 helix had no significant effect. These results demonstrated thatsome amino acid substitutions in the C34 helix, but not others, cancause a significant change in sensitivity and/or resistance toneutralization by antibodies in HIV+ sera.

The availability of the PDB (Protein Data Bank) co-ordinates of the gp41fusion domain allowed us to evaluate the impact of the substitution of Rfor Q at position 655 upon the structure and function of gp41. Using thestructure of Chan and Kim, we were able to determine that in the fusionactivated form of the gp41 trimer, Q at position 655 is located twoturns from the terminus of the C34 helix and is subject to bothintra-molecular interactions with the N36 helix of the same monomer andinter-molecular interactions with the N36 helix of adjacent monomers.The N36 and C34 helices within a gp41 monomer pack together in a fairlystandard anti-parallel coiled-coil hair-pin structure. The 3-foldsymmetric packing interface of the gp41 trimer is mediated almostexclusively by a set of parallel three-helical bundle contacts betweenthe N36 helices of each gp41 monomer. One of the few exceptions to thisis the set of contacts mediated by Q655. Although Q655 resides in theC34 helix, its side chain accepts an intramolecular hydrogen bond fromQ553 of the N36 helix within the gp41 monomer, and it donates aninter-molecular hydrogen bond to the backbone carbonyl oxygen of V551 inthe N36 helix of an adjacent gp41 monomer. The gp41 trimeric structureis thus stabilized by a “ring” of amino acids Q655-Q553-V551 in a 3-foldsymmetric repeat. Hence the three copies of Q655 contribute six hydrogenbonds that specifically stabilize the trimeric structure throughintra-molecular as well as inter-molecular hydrogen bonding contacts.Mutations of Q655 clearly possess a significant potential to disrupt thestability of the tertiary gp41 structure as well as the quaternarystructure of the gp41 trimer. Molecular modeling suggested thatreplacement of Q with R might impact the structure of the 6 coiled-coilbundle in two ways. First, the longer R side chain may have a stericeffect that disrupts the close packing of the C34 helix with the N36helix on the adjacent monomers. A second mechanism by which thissubstitution at position 655 could confer neutralization sensitivity isby disruption of the intra-molecular hydrogen bond with position 553, asthere is no longer a keto oxygen to act as a hydrogen bond acceptor.Hence the effect of the mutation is predicted to destabilize each of thegp41 monomers in the trimeric structure. However, the potential to formthe inter-molecular hydrogen bond with V551 remains, so that if the gp41monomer can still fold correctly, a partially stable trimer should beable to form.

Role of Inter- and Intra-Molecular Hydrogen Bonds.

To further investigate the role of R655 in conferring sensitivity tovirus neutralization, we used in vitro mutagenesis to replace Q atposition 655 with other residues predicted to affect inter- andintra-molecular interactions in the hydrogen bonded ring structure andexamined their affect on neutralization sensitivity (Table 2B). Some ofthe replacements, such as threonine (T), failed to yield infectiousviruses. We found that the conservative replacement of Q for asparagine(N) at position 655 resulted in a small but significant increase inneutralization sensitivity. Glutamine and asparagine share the same sidechain amide functionality, but asparagine has one fewer side chaincarbon atoms than does glutamine. Hence, the Q655N mutation is unique inthat it retains the potential to form both the intra-molecular hydrogenbond and the inter-molecular hydrogen bond, providing that a localdistortion of the helical backbone can compensate for the shortening ofthe side chain by one carbon atom. This observation explains therelative insensitivity of HIV-1 to the Q655N mutation.

We next examined the replacement of Q at position 655 with lysine (K).The side chain of lysine is shorter than that of arginine and hasreduced potential to interfere with the inter-helix packing structurethan arginine. Modeling suggested that Q655K mutation, like the Q655Rmutation, was unable to form the intra-molecular hydrogen bond withQ553, but preserved the inter-molecular hydrogen bond with V551. Wefound the Q655K mutation resulted in a highly neutralization sensitivephenotype. This result suggested that the destruction of the hydrogenbond was a more important factor in conferring neutralizationsensitivity than the steric hindrance provided by the longer side chainof arginine. This conclusion was confirmed in the next two mutantsexamined (Q655S) where serine (S) replaced glutamine at position 655,and Q655E where glutamic acid (E) replaces glutamine. We found thatthese substitutions also resulted in a significant increase inneutralization sensitivity (Table 2B), albeit not as high as the Q655Kmutation. The effect of S or E at position 655 is predicted to differfrom that of the Q655R mutation in that they preserve theintra-molecular hydrogen bond, but are unable to form theinter-molecular hydrogen bonds. Together these results suggest that boththe inter-molecular and intra-molecular hydrogen bonds are important forstabilizing the ring structure, and that disruption of either the set ofthree intra-molecular hydrogen bonds or the set of three inter-molecularhydrogen bonds results in increased sensitivity to neutralization.

Monoclonal Antibody Sensitivity and Envelope Transfer-Sensitivity toNeutralization by MAbs and Fusion Inhibitors.

While the structural analysis provided insight into the functionalconsequences of mutations at position 655, two alternate hypotheses canaccount for a mechanism by which this mutation increases sensitivity toantibody-mediated neutralization. One possibility is that this mutationis located at or near an antibody binding site and that the Q655Rmutation restores an epitope recognized by a population of neutralizingantibodies present in all four HIV-positive sera. Alternatively, it ispossible that this mutation results in a significant conformationalchange that is transmitted to other parts of gp41 such as the adjacentMPER or the gp120/gp41 trimer complex in such a way as to increaseexposure or access to antibodies at other locations on the molecule.

To explore these possibilities, antibody neutralization studies werecarried out with a panel of neutralizing MAbs to epitopes in gp120 andgp41 as well as fusion inhibitors targeting either the gp120 or the gp41portion of the HIV envelope glycoprotein. In these studies, we examinedtwo broadly gp41-neutralizing MAbs, 2F5 and 4E10; the broadlyneutralizing b12 antibody able to block CD4 binding to gp120; and 2G12,an antibody that binds to a carbohydrate epitope in gp120. In addition,we tested the antiviral entry inhibitor CD4-IgG, which binds tosequences in gp120 and is able to neutralize lab-adapted CXCR4-dependentclinical isolates at low concentrations (0.01 to 0.1 μg/ml), and primaryclinical isolates of HIV at high concentrations (10 to 100 μg/ml). Wealso examined the sensitivity of envelope mutants to enfuvirtide, apeptide virus entry inhibitor that consists of a gp41-derived peptidethat includes sequences from the C34 helix containing Q655.

The results of these studies are shown in Table 4, in which thesensitivities of clone 022 and clone 024 from subject 108060 toneutralizing MAbs were compared. It can be seen that theneutralization-resistant clone 022 is moderately sensitive to the 2F5and 4E10 MAbs specific for the MPER of gp41 but resistant toneutralization by the b12 and 2G12 MAbs reactive with gp120. This viruswas also sensitive to enfuvirtide and resistant to CD4-IgG. The highCD4-IgG concentration required for the neutralization of this virus isconsistent with the concentration required to neutralize other primary,CCR5-dependent viruses.

We next examined the neutralization-sensitive clone 024 that differsfrom the neutralization-resistant clone 022 at only seven amino acidpositions. We found that this clone was 15- to 20-fold more sensitive tothe MPER-specific MAbs (2F5 and 4E10) than the 022 clone. Similarly, theneutralization-sensitive clone 024 was more than 20-fold more sensitiveto CD4-IgG and 3.5-fold more sensitive to neutralization by enfuvirtide(Table B). Thus, clone 024 exhibited significantly increased sensitivityto neutralization by MAbs and antiviral entry inhibitors as well asantibodies in HIV-positive sera.

We then mutated the neutralization-sensitive clone 024 so as to replaceR with Q at position 655. We found that the resulting mutant (108060_024R655Q) became resistant to neutralization and showed a pattern ofneutralization sensitivity closely resembling that of theneutralization-resistant clone 022. Conversely, when we mutated theneutralization-resistant clone 022 to replace Q at position 655 with R,the resulting mutant (108060_022 Q655R), which differed from theparental neutralization-resistant clone by a single amino acid,exhibited an extraordinary increase in neutralization sensitivity (Table3). We observed a >125-fold increase in sensitivity to CD4-IgG comparedto that of the wild-type clone 022 and a 30- to 35-fold increase insensitivity to the MPER-reactive antibodies 2F5 and 4E10. We also noteda 17-fold increase in sensitivity to the antiviral drug enfuvirtide.

These results highlight the importance of glutamine at position 655 andsuggest that epistatic mutations at other sites in clone 024 moderatesensitivity to neutralization. The results of these studies areremarkable in that they show that a single amino acid substitution ingp41 not only confers sensitivity to neutralization by MAbs and entryinhibitors directed to gp41 but also increases sensitivity to CD4-IgG, amolecule that binds to gp120, an entirely different protein. Thus, theQ655R mutation appears to cause a conformational change in gp41 thataffects not only the binding of antibodies and entry inhibitors (2F5,4E10, and enfuvirtide) that bind close to the site of the mutation butalso the binding of another inhibitor (CD4-IgG) that binds to a site ongp120 located a considerable distance from the mutation.

Transfer of the Q655R Mutation to Related and Unrelated Viruses.

In order to determine whether the Q655R mutation could conferneutralization sensitivity and resistance to other viruses, thismutation was introduced into two unrelated viruses highly resistant toneutralization (from subjects 108069 and 108051) that normally possesseda Q at a position corresponding to 655 of the virus from subject 108060(the 108060 virus). The results of these experiments are shown in Table3. Interestingly, we found that the replacement of Q655 with R hadlittle or no effect on neutralization by any of the HIV-positive sera.However, these mutations significantly increased the sensitivity toneutralization by the 2F5 and 4E10 MAbs (25- to 35-fold). Thesemutations also increased the sensitivities to neutralization by theentry inhibitors enfuvirtide and CD4-IgG. Thus, the mutation of Q to Rat a position corresponding to 655 in the 108069 virus increased thesensitivity to enfuvirtide by more than 17-fold and increased thesensitivity to CD4-IgG by more than 20-fold. The 108069 mutant with theQ655R mutation seemed to be somewhat more sensitive to enfuvirtide andpossibly CD4-IgG than the corresponding mutant of the 108051 virus.

Together, these results demonstrate that the mutation of Q to R atpositions corresponding to 655 of the 108060 virus confers sensitivityto neutralizing MAbs to the MPER and antiviral compounds targeted to theC34 helix and the MPER of gp41. However, it was interesting that thesemutations failed to increase the sensitivity to bNAbs in HIV-positivesera. We do not know whether neutralizing activity in HIV-positive serais attributable to antibodies binding to the C34 region, the MPER, orother parts of the molecule. It has been recently reported thatantibodies with specificities similar to 2F5 and 4E10 are rare inHIV-positive sera, which might account for the lack of effect.Alternatively, the failure of the Q655R mutation to increaseneutralization sensitivity by HIV-positive sera might be attributable topolymorphisms outside of the MPER and the C34 region that preclude thebinding of otherwise bNAbs. This may well be the case since the 108069and 108051 viruses were selected because of their resistance toneutralization by the HIV-positive sera selected for use in thesestudies.

Transfer of Q655R mutation to related and unrelated viruses. In order todetermine whether the Q655R mutation could confer neutralizationsensitivity and resistance to other viruses, this mutation wasintroduced into two unrelated viruses (108069 and 108051) that normallypossessed a Q at a position corresponding to 655 of the 108060 virus.The results of these experiments are shown in Table 3. Interestingly, wefound that replacement of Q655 with R had little or no effect onneutralization by any of the HIV+ sera (supplementary information S4).However, these mutations significantly increased the sensitivity toneutralization by the 2F5 and 4E10 MAbs (25- to 35-fold). Thesemutations also increased sensitivity to neutralization by the entryinhibitors FUZEON and CD4-IgG. Thus the mutation of Q to R at a positioncorresponding to 655 in 108069 increased the sensitivity to FUZEON bymore than 17-fold and increased the sensitivity to CD4-IgG by more than20-fold. The 108069 mutant with the Q655R mutation seemed to be somewhatmore sensitive to FUZEON and possibly CD4-IgG than the correspondingmutant in the 108051 virus. Together these results demonstrate that themutation of Q to R at positions corresponding to 655 of 108060 conferssensitivity to neutralizing MAbs and anti-viral compounds targeted tothe C34 and MPER regions of gp41 and to the CD4 binding site in gp120.However, this mutation is not able to confer sensitivity toneutralization by bNAbs in HIV+ sera to all viruses.

Discussion

These studies utilized a novel method for the identification and mappingof mutations that affect the sensitivity/resistance of viruses toneutralization by HIV+ sera and anti-viral entry inhibitors. Thisapproach differs from previously described methods of mutationalanalysis used to study HIV in that it relies on naturally occurringmutations in the swarm of closely-related viruses that evolve during thecourse of HIV infection.

Identification of a mutation at position 655 in gp41 that conferssensitivity to neutralization by bNAbs. In this study we identified anaturally occurring mutation (Q655R) that affects sensitivity/resistanceof viruses to neutralization by bNAbs. X-ray crystallography studiesshowed that glutamine at position 655 is located close to the C-terminusof the C34 helix and contributes to two hydrogen bonds: one mediating anintra-molecular interaction with the N36 helix on the same monomer, andthe other mediating an inter-molecular interaction with the N36 helix onan adjacent monomer. These two hydrogen bonds appear to stabilize thefusion active conformation of the 6 helix bundle in trimeric gp41 insuch a way as to increase infectivity and confer resistance toneutralization. Our data suggest that naturally occurring mutations(e.g. Q655R) and experimental mutations (e.g. Q655K, Q655S or Q655E)that interfere with either the intra-molecular or inter-molecularhydrogen bonds normally provided by Q655 confer sensitivity toneutralization by interfering with the formation of the hydrogen bondedring. In this regard, the function of this ring structure appears to betwofold: 1) to stabilize interactions between the backbones of adjacentN-36 helices in the core of the 6 helix bundle and 2) to stabilize theends of the coiled-coil hairpin structures in each gp41 monomer. Thislatter interaction may serve a function analogous to the fibular claspon brooch or badge.

The mechanism by which the Q655R mutation confers sensitivity andresistance to neutralization. HIV fusion is thought to be a step-wiseprocess that begins with the binding of CD4 and a suitable chemokinereceptor (CXCR4 or CCR5) to gp120. This triggers a conformational changeresulting in the formation of the “pre-hairpin” fusion intermediatecomplex via rearrangement of the amphipathic helices in the externaldomains of gp41. The N36 helices pack in a parallel three-helicalbundle. The pre-hairpin is characterized by the exposure of theN-terminal hydrophobic fusion domain and the C-terminal MPER of gp41which are normally folded inside the gp41 trimer and not exposed tocirculating antibodies. Further molecular rearrangements result inclosure of the hairpin structure, resulting in anti-parallel packing ofeach C34 helix into the grooves on the outside of each N-helix in thegp41 trimer. Ultimately, a highly thermostable 6 helix bundle is formed,which is thought to provide the energy required to fuse viral withcellular membranes. We hypothesize that the Q655R mutation alters theconformational equilibria so as to favor the pre-hairpin fusionintermediate structure where both the N terminal fusion domain and MPERare exposed. This would explain the increased sensitivity to the 2F5 and4E10 MAbs, which recognize the exposed MPER as well as the increasedsensitivity to neutralization by CD4-IgG. Previous studies havesuggested that transition of the fusion intermediate to thefusion-active conformation is the rate limiting step in virus infection,and is estimated to be in the range of 15 minutes based on T-20 (FUZEON)sensitivity. An interesting possibility is that HIV envelopeglycoproteins that are “trapped” into the fusion intermediateconformation might represent superior HIV vaccine antigens, since theywould expose epitopes normally hidden, and only exposed during virusfusion. The results obtained with swarm analysis are consistent with thepossibility that mutations at 655 in the 108060 virus, such as Q655R andQ655K, alter the conformational equilibria to favor the gp41 trimer inthe fusion intermediate conformation.

Sera from early infections may represent an opportunity to identify raremutations that confer sensitivity to bNAbs. Based on the examination ofsequence data in the Los Alamos HIV Sequence database, it appears thatthe mutation of arginine for glutamine at position 655 is extremely rareand occurs with an observed frequency of 8/1242 (0.64%). How is it thenthat we were able to find such a rare mutation within the first sevenviruses examined? One possible explanation relates to the fact that theviruses analyzed in this study were all collected close to the time ofinfection, and may possess antigenic structures that are uncommon inviruses recovered from later infections due to kinetics of thedevelopment of the neutralizing antibody response. Several studies haveshown that bNAbs do not occur until 6-12 months after infection. Itcould well be the case that viruses recovered from early infectionspossess a broader range of antigenic features because they are beingselected primarily for infectivity rather than neutralizationresistance. Once effective neutralizing antibodies are present,neutralization sensitive variants, such as Q655R, would be selectedagainst, and rapidly disappear from plasma. The possibility that virusesfrom early infections may contain mutations resulting in unusualstructures is consistent with a previous study where viruses recoveredfrom the same clinical cohort as 108060 had an unexpectedly highfrequency of mutations that affected the disulfide structure of gp120.

Envelope proteins from early infections with rare mutations such asQ655R may represent a new source of vaccine antigens. How are mutationsthat occur with such low frequencies useful for HIV vaccine development?The results obtained for the Q655R mutation suggest that mutations ofthis type significantly alter the antigenic structure of the envelopeprotein in such a way as to expose important epitopes that are normallyshielded from contact with the immune system. Frey et al. havehypothesized that immunization with a gp41 trimer locked into thepre-hairpin fusion intermediate conformation might be an effective wayto elicit bNAbs to the MPER with activities similar to 2F5 and 4E10. Webelieve that the Q655R and other mutations that we have described mayhave “trapped” the gp41 trimer into this pre-hairpin intermediateconformation and might be effective in inducing bNAbs. Theimmunogenicity of such variants has not yet been explored; however,studies are underway to examine their immunogenic potential.

Virus fusion is a delicately balanced process that involves majorconformational transitions triggered by ligand binding. Thesetransitions are no doubt aided, and stabilized, by a variety ofcooperative interactions. The studies described highlight a set of novelinteractions mediated by hydrogen bonds that appear to facilitate fusionof viruses with cellular membranes. The 6 helix bundle structure andfusion mechanism is conserved throughout evolution and is essential forthe infectivity of most enveloped viruses. A homologous 4 helix bundleplays a similar role in cellular vesicles mediating intracellulartransport and secretion. It may well be that the infectivity of otherenveloped viruses (e.g. influenza) as well as membrane fusion processes(e.g. intracellular transport and secretion) might also depend onstabilizing interactions from hydrogen bonded structures of the typethat we have observed in gp41. Knowledge of these stabilizinginteractions may be useful in understanding the details of the fusionprocess and may provide a new approach to the development of vaccine andtherapeutic products, where alteration of these interactions may providea functional benefit.

Materials and Methods

Sera and Plasma. Cryopreserved plasma used to clone full length envelopeglycoproteins were collected in the course of a Phase 3 clinical trialof a candidate HIV vaccine (AIDSVAX B/B) sponsored by VaxGen, Inc. (S.San Francisco, Calif.). Deidentified specimens and data required forthese studies were provided by Global Solutions for Infectious Diseases(S. San Francisco, Calif.). All of the viruses used in this study wereobtained from patient plasma collected within six months of initialinfection. HIV+ sera containing broadly neutralizing antibodies (Z23,Z1679, Z1684, and N16) were provided by Monogram Biosciences, Inc. (S.San Francisco, Calif.) and are known from previous studies to neutralizea variety of primary clinical isolates of HIV. The monoclonal antibodiesused in these studies were obtained from two different sources. Thebroadly neutralizing monoclonal antibodies b12, 2F5, and 4E10 wereobtained from the NIH AIDS Reagent Repository and Polymun A.G. (Vienna,Austria). The antiviral compound CD4-IgG was described previously andprovided by GSID (S. San Francisco, Calif.).

Construction of envelope gene libraries and pseudoviruses. Libraries ofenvelope glycoprotein were created from each subject by PCRamplification of full length envelope genes from cryopreserved plasmausing the method described previously. The swarm of PCR products wascloned into a plasmid expression vector useful for the construction ofpseudoviruses. The vector was specifically designed to permit theconstruction of pseudovirus libraries for use in a well-established andvalidated virus neutralization assay. However, instead of pooling all ofthe clones together and carrying out neutralization assays or drugsensitivity assays with an entire library of cloned genes from eachinfected individual as had been done previously, we plated out theplasmid library on agar plates and picked 24-48 clones from eachindividual for infectivity studies. The plasmid DNA was isolated fromeach clone and used to create a stock of pseudovirus particles that werethen screened for infectivity, and chemokine receptor usage. Afterverifying infectivity and receptor usage, we then selected approximatelyten CCR5-dependent pseudotype viruses with good infectivity for virusneutralization assays. The virus neutralization assays were carried outas described by Schweighardt et al.

Sequencing and mutagenesis. Plasmids containing cloned envelopeglycoproteins were sequenced using fluorescently labeleddideoxynucleotides at either Monogram Biosciences or the University ofCalifornia Sequencing Facility (Berkeley, Calif.) using capillaryelectrophoresis sequencing devices (Applied Biosystems, Foster City,Calif.). HIV envelope glycoprotein sequences were mutagenized by amismatched primer method using the QuikChange Mutagenesis kit(Stratagene, San Diego). All mutations were confirmed by DNA sequencing.The numbering of amino acids is made with reference to the sequence ofgp160 from clone 022 of from subject 108060. Position 655 corresponds toposition 653 of the HXB2 reference strain of HIV-1.

Virus neutralization assay. The automated virus neutralization assaydescribed in this study has been described previously. Theneutralization data reported represent IC50 values calculated from serumdilution curves. This assay employs multiple assay controls, including apositive pseudotype virus control panel and a negative pseudotype viruscontrol panel. Assay acceptability criteria have been established tominimize interassay variability and assure comparability of data fromdifferent experiments. The positive virus control panel includes thepseudotypes from the neutralization sensitive isolate, NL43, and theless neutralization sensitive primary isolate JRcsf. The negative virus(specificity) control consists of pseudotype viruses prepared from theenvelope of the amphitropic murine leukemia virus. Previous studies(Wrinn, Montefiore, and Sinangil, manuscript in preparation) have shownthat the Monogram virus neutralization assay yields comparable resultsto the TZM-BL pseudotype virus neutralization assay when tested onstandard panels of HIV-1 isolates distributed by the NIH.

Molecular modeling. Although the complete gp41 HIV-1 glycoproteinstructure is currently unavailable, a crystal structure comprising theN36 and C34 helices of gp41 (PDB accession code 1AIK) anti-parallelhelical core duplicates the essential intra-molecular as well asinter-molecular packing interactions in which the crystallographicthree-fold axis corresponds to the natural gp41 trimer three-fold axis.The intra-molecular and inter-molecular hydrogen bonding contactsinvolving Q655 were identified in the context of the gp41 trimericstructure in the PyMOL molecular visualization software package. Thepotential effects of the various Q655 mutations upon both sets ofpacking interactions were then analyzed by in silico mutagenesis inPyMOL combined with crystallographic symmetry-constrained energyminimization molecular modeling (using the crystallographic softwarepackage Phenix to enforce the gp41 trimeric symmetry. The results of thecrystal-structure-based molecular modelling data were subsequentlyanalyzed in PyMOL.

Detailed Description of the Embodiments Related to Selection of HIVVaccine Antigens by Use of Intrapatient Sequence Variation to IdentifyMutations in the HIV Envelope Glycoprotein that Affect the Binding ofBroadly Neutralizing Antibodies (See U.S. Provisional Application61/195,112 Filed 4 Oct. 2008 and International Application No.PCT/US09/59583 Filed 5 Oct. 2009).

Disclosed is a new method for identifying mutations in envelopeproteins, which methods comprise analyzing intra-patient HIV-1 virusvariation to identify specific amino acid residues of the HIV-1 envelopeglycoproteins, gp160, gp120, and gp41 that affect sensitivity orresistance to broadly neutralizing HIV-1 antibodies. The mutationsidentified by the methods of the invention provide enhanced sensitivity(or resistance) to neutralization of a virus by anti-viral antisera; inparticular neutralization of an HIV virus by anti-HIV antibodies, suchas in antisera. The methods described identify epitopes recognized bybroadly neutralizing antibodies. Such epitopes and the proteins of whichthey are a part may provide a powerfully immunogenic, protective vaccineagainst HIV. To identify polymorphisms and sequences that effectsensitivity or resistance to broadly neutralizing antibodies, viralenvelope sequences (such as gp160, gp120, and gp41) from sensitive andresistant viruses were identified and compared and the differences werenoted. Mutagenesis was carried out to identify specific residues thatcorrelated with sensitivity or resistance to virus neutralization.

Essentially, the method consists of carrying out the following steps:(i) Providing a plurality of individual subjects who are seropositivefor HIV antibodies and taking a biological sample such as blood orplasma from each subject, wherein the sample contains a multiplicity ofHIV viruses with closely related genomes, wherein all subjects had beeninfected with HIV no more than one year before, and no less than onemonth before sample collection. (ii) Amplifying the env genes by thepolymerase chain reaction (PCR) of the multiplicity of viruses toproduce a library of different env genes. (iii) Cloning the amplifiedenv genes into a plasmid shuttle vector allowing the plasmid toreplicate in both bacteria (such as E. coli) and mammalian cells. Suchvectors contain: a bacterial origin of replication, an origin ofreplication from a mammalian cell virus such as SV-40 or adenovirus, anda functional transcription unit that enables expression of a suitabledrug resistance gene such as ampicillin, tetracycline, or kanamycin inorder to allow selective growth of bacteria transformed with the shuttlevector. The shuttle vector must also contain the elements of afunctional mammalian cell transcription unit. Beginning at the 5′ end ofthe sense DNA strand, the transcription unit should contain a promotersequence from a mammalian gene or virus, a splice donor/acceptor site, asegment of synthetic DNA containing either multiple restriction enzymerecognition sites or other sequences to allow directional cloning of PCRamplified envelope genes, a transcription termination codon, and apolyadenylation site. The transcription unit should also containtranscription enhancer sequences at either locater either 5′ to thepromoter or 3′ of the polyadenylation site. Once PCR amplified HIV genesare ligated into the shuttle vector, the collection of plasmidscontaining the cloned envelope genes are transformed into E. coli bystandard techniques, grown in a small volume of bacterial culture mediaand then plated onto agar plates containing the appropriate antibioticso that only bacterial containing the shuttle vector plasmid containingthe cloned envelope genes are able to form colonies. Individual coloniesare then selected at random and plasmid DNA from each colony is preparedand analyzed by restriction digestion, and only those containing aninsert of the proper size of the full length HIV envelope gene areretained and used for the preparation of pseudoviruses as describedbelow. (iv) Co-transfecting mammalian cells (e.g. 293HEK) with theenv-containing vector and simultaneously with a plasmid containing adefective HIV provirus virus where the coding sequence of the env genewas replaced with the coding sequence of a marker gene such as onecapable of emitting light, e.g. Luciferase) to produce pseudovirionscontaining the amplified env genes. (v) The pseudovirions are placed incontact with cells capable of being infected by HIV so as to producecolonies of infected cells. Such cells express the genes for CD4 and atleast one chemokine receptor gene (either CCR5 or CXCR4). The cells canalso express CD4 and both the CCR5 and CXCR4 chemokine receptor genes.Cell culture supernatants containing pseudoviruses are harvested fromthe transfected cells and individual stocks of pseudoviruses resultingfrom single purified expression plasmids represent virus stocks. (vi)The pseudotype virus colonies thus created are tested to determineinfectivity; 20-50 pseudo virus stock are prepared from each individualand only those exhibiting good infectivity as measured by a significanthigher level of relative light units relative to control pseudovirusescontaining only defective envelope genes are advanced to neutralizationassays. (vii) Then each infective pseudotype virus is tested forsensitivity or resistance to neutralization by one or more broadneutralizing antibodies. In neutralization assays two or morepseudovirions from the same individual are tested. Each pseudovirusstock is incubated with serially diluted plasma or sera from HIVinfected individuals or purified polyclonal or monoclonal antibodies. Asignificant decrease in the emission of light relative to pseudovirusesincubated with a negative control specimen that does not containantibodies to HIV envelope proteins. (viii) Then selection is done ofpairs of plasmids containing specific env proteins which were used toprepare the pseudoviruses described above, wherein each pair containsone env gene that yielded a neutralization resistant pseudovirus and oneenv gene that yielded neutralization sensitive pseudovirion. (ix) Theenvelope genes from sensitive and resistant pseudoviruses are thensequenced and comparison was done to thus to identify amino acidsequence differences between the neutralization sensitive andneutralization resistant envelope genes. Only pairs of sequences with aminimal number of sequence differences (no more than for example 10%,8%, 6%, 5% or 4% sequence difference over the entire coding region ofthe env sequence in question) are then selected for further analysis.(x) In vitro mutagenesis may then be performed to create envelope geneswhere the effect of each amino acid difference between theneutralization sensitive and neutralization resistant pairs can bedetermined when such mutant genes are incorporated into pseudovirionsand tested for sensitivity and resistance to neutralization. In thisstep, amino acids at corresponding positions of neutralization sensitivemember of the pair is introduced into the neutralization resistantmember of the pair to see if it confers the neutralization sensitivephenotype. Conversely, specific amino acids from the neutralizationresistant sequence can be introduced into the neutralization sensitiveenvelope gene by in vitro mutagenesis to identification of the specificamino acid responsible for the neutralization resistant phenotype.

It should be noted that it is an important feature of the invention thatthe samples be taken from individuals within a certain window. Forvarious reasons more thoroughly explained elsewhere in this disclosure,the HIV virus population changes dramatically during the course ofinfection, and the inventors have reasoned that in order to successfullyidentify the polymorphisms of the invention, samples need to be takenwithin a certain window of time. In the present invention samples needto be taken from subjects who had been infected with HIV no more thanone year before, and no less than one month before sample collection. Invarious embodiments a wider window may be used and samples may be takenno more than 18 months before, and no less than two weeks before samplecollection. In other embodiments a narrower window may be used and theearliest and latest times that bracket the sample window may be, forexample, 14 months and 1 month, 12 months and 1 month, 10 months and 6weeks, 8 months and 6 weeks, 6 months and 6 weeks, or any combination ofthese times from the date of infection. Obviously the date of infectionis not always precisely known, and the dates that comprise the earliestand latest times since infection may vary, for example +/−14 days or+/−24 days. In one specific embodiment used to produce the currentexperimental results, all subjects had been infected with HIV 109days+/−58 days before specimen collection.

Although most of the viruses from an individual exhibited a predominant“neutralization sensitive” or “neutralization resistant” phenotype,variants were identified that differed in sensitivity from predominantforms. Because all of the samples compared were from recent infectionsthe amount of intra-patient sequence variation in the envelopeglycoprotein was minimal. Site directed mutagenesis enabled us toidentify amino acids residues responsible for neutralization sensitivityor resistance. Mutations affecting virus neutralization were found inboth gp120 and gp41.

The methods disclosed provide a novel strategy to enable quick andefficient identification of the epitopes recognized by bNAbs in HIV+patient sera. Characterization of polymorphism at these sites willprovide information to guide the formulation of multivalent vaccines. Inone aspect, the invention discloses methods for identification ofcertain immunogenic epitopes, and further discloses the epitopesthemselves. Broadly neutralizing antibodies recognize the specificepitopes of the HIV-1 envelope glycoproteins, including gp120, and gp41and any gp160-derived protein, whether monomeric or oligomeric. Thus,aspects of the present invention include these HIV-1 envelopeglycoproteins, nucleic acids encoding the polypeptides and vaccinescomprising the polypeptides or nucleic acids.

Also described are methods for the identification of specificpolymorphisms within, or having an effect upon, neutralizing epitopesthat are suitable for inclusion in a protein or polypeptide that may beincluded in the formulation of a multivalent HIV vaccine cocktail. Itshould be noted that the polymorphisms of the invention need not bewithin or even close to the epitopes affected. The polymorphisms of theinvention alter the conformation of the epitopes so as to reveal (orhide) a portion of the epitope in such a way that it becomes availableto bind with (or hidden from) a corresponding antibody, such as abroadly neutralizing antibody. Further described is a method foridentifying and purifying broadly neutralizing antibodies from HIVpatient serum or plasma. HIV envelope genes were amplified from HIV+plasma obtained in the VAX004 Phase 3 trial. See Flynn, N. M., D. N.Forthal, C. D. Harro, F. N. Judson, K. H. Mayer, and M. F. Para. 2005.Placebo-controlled phase 3 trial of a recombinant glycoprotein 120vaccine to prevent HIV-1 infection. J Infect Dis 191:654-65.

Also disclosed are vectors, pseudoviruses and other constructs thatcomprise specific polynucleotide sequences and mutations that encodeantigens and epitopes described. Also disclosed are generic and specificsequences, polymorphisms, mutations, antigens and epitopes that may beused for the treatment and/or prevention of viral infection such as HIVinfection.

Also disclosed are medicaments and therapeutic formulations such asvaccines that comprise antigens and epitopes of the invention or thatcomprise polynucleotide sequences or vectors encoding antigens andepitopes of the invention. Vaccines of the invention may be used both totreat an infection once the infection has occurred, so as to prevent orcure a disease, and more commonly, to prevent an infection. Alsodisclosed are therapeutic methods that comprise delivering a vaccine toa subject wherein the vaccine may comprise one or more antigens orepitopes of the invention, or polynucleotide sequences or vectorsencoding antigens and epitopes of the invention. Also described arespecific glycoproteins, polypeptides, proteins and epitopes which may beformulated as part of an effective vaccine. Also described arepolyclonal and/or monoclonal antibodies that may be used as therapeuticagents for passive immunization. The vaccines of the invention may beprotein/polypeptide antigen vaccines, or may be polynucleotide vaccineswherein the polynucleotides express antigenic proteins that provoke aprotective immune response.

Also disclosed are therapeutic methods that employ compositions such asdrugs and small molecules or antibodies that interact with specificantigens or epitopes or regions of the glycoproteins or polypeptidesdescribed, thereby (i) exposing a previously unexposed epitope whichepitope can bind specifically with a neutralizing antibody and/or (ii)limiting, inhibiting or preventing fusion of a viral membrane with acell membrane, thereby inhibiting infection of a call by a virus. Alsodisclosed are the therapeutic compositions, drugs, small molecules orantibodies used in the above method.

Also described are compositions containing specific sequences and aminoacid substitutions, deletions and additions that affect the confirmationof a protein or a polypeptide so as to hide or expose one or moreparticular epitope. Also described are methods of contacting a viruswith such a composition to affect the confirmation of a protein or apolypeptide so as to hide or expose one or more particular epitope so asto expose a previously unexposed epitope which epitope can bindspecifically with a neutralizing antibody and/or to limit, inhibit orprevent fusion of a viral membrane with a cell membrane.

Also described are polypeptides containing the epitopes of theinvention, nucleic acids encoding the polypeptides, vaccines comprisingthe polypeptides or nucleic acids, and methods of attenuating orpreventing HIV infection via administration of the vaccines.

Also described are nucleic acids encoding the polypeptides of theinvention and vectors that comprise nucleic acids encoding thepolypeptides of the invention, which vectors may be used for therapeuticand/or vaccination purposes.

Further, the invention isolated polynucleotides encoding thepolypeptides of the invention, a polypeptide comprising a) an amino acidsequence selected from any sequence described herein, b) an amino acidsequence having at least 90% sequence identity to an amino acid sequencedescribed herein, c) a biologically active or immunogenic fragment of anamino acid sequence described herein. The invention further provides anisolated polynucleotide comprising a polynucleotide sequence having atleast 90% sequence identity to a polynucleotide described, or apolynucleotide sequence complementary to the foregoing. In onealternative, the polynucleotide comprises at least 60 contiguousnucleotides. The invention also includes any of the polypeptides encodedby such polynucleotides. Additionally, the invention provides anisolated antibody which specifically binds to an amino acid sequencedescribed herein.

The investigators have identified various specific polynucleotide andpolypeptide envelope sequences that contain specific polymorphisms suchas a substitution of arginine for glutamine at position 655 in gp41(“Q655R”). The invention includes these sequences and also encompassesother similar and related sequences that display the same specificpolymorphism at a location identifiable as being homologous to Q655R inthe HIV env gene as disclosed in SEQ ID NO:16.

To say that a first particular sequence of amino acids, or a particularsingle amino acid residue or polymorphism “corresponds to” a particular(second) sequence, site or position on a known sequence means that thefirst sequence, residue or polymorphism is located at a position that isreadily identifiable by virtue of sequence homology as being equivalentto a known sequence, site or position on a known sequence on the second,known sequence. The same reasoning may be applied to polynucleotides.

To say that a first particular sequence or specific polymorphism is“identifiable as being homologous to” a second particular sequence orpolymorphism means that the sequences shows homology or sequenceidentity with each other so as to be identifiable as being homologues(and quite possibly, paralogs) of the same gene. Such homology isusually evident to one of skill in the art and can be determined by eye.Additionally various algorithms such as BLAST may be used.

In the present case, the region in which the polymorphism is found ishighly conserved between variants, and the recognition of sequences orpolymorphisms as being located at a site “identifiable as beinghomologous to” amino acid 655 in SEQ ID NO:16 is clear and easilyunderstood. In the present case the invention includes a substitution ofQ to another residue such as R at a site identifiable as beinghomologous to amino acid 655 in SEQ ID NO:16.

The env polypeptide may be selected from any of the known env sequences,or may be a previously unpublished sequence having a certain degree ofsequence similarity to one of the known env sequences.

For example, the env polypeptide of the invention may comprise asequence with a substitution of arginine for glutamine at positionidentifiable as homologous to position 655 within in a gp41, wherein theenv polypeptide has at least 60% identity (or, in other embodiments, atleast 70%, at least 80%, or at least 87% or at least 90% or at least 95%or at least 98% or at least 99% identity) using BLASTP 2.2.21 withdefault settings (see Altschul et al., (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402) to one of the following sequences: SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20.

Alternatively, for example, the env polypeptide of the invention maycomprise a sequence with a substitution of arginine for glutamine atposition identifiable as homologous to position 655 within in a gp41,wherein the env polypeptide has at least 65% identity (or at least 70%,80%, 87%, 90%, 95%, 98% or at least 99% identity) using BLASTP 2.2.21with default settings to one of the following sequences described inthis application as: p1.10848_c2 Resistant, p1.10848_c11 Sensitive,108051_c6 Sensitive, p1.108051_c5 Resistant, p1.108060_c22 Resistant, orp1.108060_c24 Sensitive.

Any of the above sequences may additionally include signal sequences ofvariable length or sequences that assist trimer at either the 5′ or 3′ends. Any of the above sequences may be truncated by deletion ofsequences encoding the transmembrane domain and cytoplasmic tail of thegp41 region of the gp160 gene.

Any of the above sequences may also be expressed as a fusion proteinwhere nucleotides encoding the signal sequence and 0-12 N-terminalresidues of the mature HIV envelope protein are deleted from the HIVenvelope gene and replaced by nucleotide sequences encoding the signalsequence from another highly expressed protein to facilitate expressionin mammalian cells. Examples of suitable signal sequence include thoseof herpes simplex virus 1 glycoprotein D or the prepro signal sequenceof human tissue plasminogen activator. It is also sometimes desirable toinclude nucleotide sequences encoding a flag epitope immediatelyadjacent to the signal peptidase cleavage site at the N-terminus of themature gp140 protein, or a flag epitope adjacent to the C-terminalsequence of the gp140 protein to facilitate purification. The flagepitope can be any 4-30 amino acid sequence recognized by a monoclonalantibody suitable for immunoaffinity chromatography, or can be a clusterof amino acids such as a poly-histidine (his-tag) sequence that canmediate adherence to an insoluble matrix for affinity purification. Inthis regard it is important that a simple, non-denaturing process isavailable to elute the poly-histidine fusion containing fusion proteinform the insoluble matrix. In some cases (e.g. herpes simplex virusglycoprotein D) the flag epitope can be derived from the same protein asthe heterologous signal sequence. The flag epitope can be attached toany amino acid within the first 20 amino acids of the gp120 portion ofthe molecule. An example of this is fusion adjacent to the conserved Vat position 41 within the full length gp160 sequence and located at thesequence beginning VPVWKEA (SEQ ID NO:21). Amino acid residuescorresponding to a heterologous flag epitopes can be located either atthe amino terminus of the mature protein.

Glycoprotein gp140 may be expressed as a fusion protein lacking thefurin cleavage site. In another embodiment, it may be necessary tomutagenize the highly conserved furin cleavage site that occurs at thejunction between gp120 and gp41 in order to insure that the gp41 domainis covalently attached to the gp120 domain during purification andpossibly during immunization.

Glycoprotein gp140 may include sequences attached at the C-terminus ofgp140 to facilitate oligomerization into gp140 trimers. In order tocreate an antigen that replicates the structure of the HIV envelopeprotein on the surface of virions, it is often desirable to producegp140 trimers. To accomplish this goal, one can use one of the severalstrategies such as the addition of a GCN4 coiled coil domain or the T4fibrin tag that have been described and successfully used by otherinvestigators to produce stable gp140 trimers. Locations where sequencescould be attached are within 7 amino acids of the C terminus of gp140 asindicated. Thus, for example, the invention includes a compositioncomprising a purified HIV env polypeptide, the polypeptide having aQ655R substitution, and having at least 90% amino acid sequence identityto one of the following sequences: SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20. Such compositions include vaccines.Additionally, the invention encompasses an isolated antibody whichspecifically binds to a purified HIV env polypeptide, the polypeptidehaving a Q655R substitution, and having at least 90% amino acid sequenceidentity to one of the following sequences: SEQ ID NO: 16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20. Vaccines of the presentinvention can be used in a prophylactic manner to prevent HIV infectionor in a passive therapeutic manner to attenuate existing HIV infection.Vaccines of the present invention may be multivalent, i.e., containmultiple HIV antigens, for example, containing two more HIV-1 envelopeglycoproteins, gp160, gp120, and gp41 which present one of more epitopesthat bind specifically to broadly neutralizing antibodies. Vaccines ofthis invention may be administered alone or in combination with otherHIV antigens and/or adjuvants, cofactors or carriers. The HIV1 envelopeprotein or nucleic acid may be administered in combination with otherantigens in a single inoculation “cocktail”. Adequacy of the vaccinationis determined by assaying antibody titers or the presence of T cellsand/or the viral load may be monitored. The polypeptides of thisinvention may optionally be administered along with other pharmacologicagents used to treat AIDS or ARC or other HIV-related diseases andinfections, such as AZT, CD4, antibiotics, immunomodulators such asinterferon, anti-inflammatory agents, and anti-tumor agents.

The invention also encompasses constructs containing the sequence ofgp160, gp140 or gp41 from neutralization resistant clone 22 from subject108060 in which a mutation is present, the mutation (Q655R) created byreplacement of glutamine with arginine at position 655. The mutation maybe introduced by standard in vitro mutagenesis techniques. Note that thebasic gp160 sequence (prior to the Q655R mutation) is that from aneutralization resistant clone, and not the neutralization sensitiveclone. The Q665R neutralization resistant sequence appears to be moreimmunogenic than the Q665R neutralization sensitive sequence and confersa stronger neutralizing and protective antibody response. This is notwhat would have been predicted.

Possible preferred embodiments include constructs containing thesequences of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, andSEQ ID NO:20 described herein.

SEQ ID NO:16 is the full length gp160 854 residue sequence (fromp1.108060_c22) with the Q655R mutation.

SEQ ID NO: 17 is a truncated form of the envelope protein lacking thegp41 transmembrane domain and cytoplasmic tail, termed gp140. In thisembodiment the gp160 gene is truncated by deletion of sequences encodingthe transmembrane domain and cytoplasmic tail of the gp41 region of thegp160 gene. This is accomplished by introduction of a stop codon (e.g.TAA) after any of the amino acids in the following sequence locatedadjacent to the start of the gp41 transmembrane domain: SWLWYIK (SEQ IDNO:39).

SEQ ID NO:18 is a fusion protein where the signal sequence of HIV hasbeen deleted and replaced with the signal sequence of another highlyexpressed protein. The fusion protein is designed to facilitateexpression in mammalian cells, and is termed gp140-FP. This embodimentincludes at least 95% of gp120 and the extracellular domain of gp41. Itspecifically lacks the transmembrane domain and cytoplasmic tail ofgp41. The molecule is best expressed as a fusion protein wherenucleotides encoding the signal sequence and 0-12 N-terminal residues ofthe mature HIV envelope protein are deleted from the HIV envelope geneand replaced by nucleotide sequences encoding the signal sequence fromanother highly expressed protein to facilitate expression in mammaliancells. Examples of suitable signal sequence include those of herpessimplex virus 1 glycoprotein D or the prepro signal sequence of humantissue plasminogen activator. It is also desirable to include nucleotidesequences encoding a flag epitope immediately adjacent to the signalpeptidase cleavage site at the N-terminus of the mature gp140 protein,or a flag epitope adjacent to the C-terminal sequence of the gp140protein to facilitate purification. The flag epitope can be any 4-30amino acid sequence recognized by a monoclonal antibody suitable forimmunoaffinity chromatography, or can be a cluster of amino acids suchas a poly-histidine (his-tag) sequence that can mediate adherence to aninsoluble matrix for affinity purification. In this regard it isimportant that a simple, non-denaturing process is available to elutethe poly-histidine fusion containing fusion protein form the insolublematrix. In some cases (e.g. herpes simplex virus glycoprotein D) theflag epitope can be derived from the same protein as the heterologoussignal sequence. The flag epitope can be attached to any amino acidwithin the first 20 amino acids of the gp120 portion of the molecule. Anexample of this is fusion adjacent to the conserved V at position 41within the full length gp160 sequence and located at the sequencebeginning VPVWKEA (SEQ ID NO:21). Amino acid residues corresponding to aheterologous flag epitopes can be located either at the amino terminusof the mature protein.

(SEQ ID NO:19) is a gp140 from 108060_c22 Q655R containing gp120 and theextracellular domain of gp41 with Q655R mutation expressed as a fusionprotein and lacking the furin cleavage site.

(SEQ ID NO:20) is a gp140 from 108060_c22 Q655R containing gp120 and theextracellular domain of gp41 with Q655R mutation expressed as a fusionprotein and containing sequences to facilitate or stabilize trimerformation.

Experimental Procedures, Materials, Methods and Results

Described is a new method to identify the epitopes recognized by broadlyneutralizing antibodies by taking advantage of the naturally occurringamino acid sequence variation (intra-patient variation) that evolveswithin every HIV-infected individual. This method also allows one todefine molecular determinants of sensitivity and resistance to antibodymediated neutralization, and allows for the design of a new class ofantiviral drugs. We have used this method to identify a mutation in theHIV fusion protein, gp41, that markedly affects sensitivity andresistance of primary HIV-1 isolates to neutralization by HIV+ sera. Thenew approach that we describe provides a powerful and convenient methodto identify epitopes recognized by bNAbs in HIV+ sera and will enablethe development of new immunogens that target these sites.

Studies of the early events in infection have shown that transmission ofHIV-1 involves a genetic bottleneck where, out of the myriad of geneticvariants in each HIV infected donor, only a single homogeneous variantof HIV-1 successfully replicates in the recipient. This variantreplicates to very high titers for the first days and weeks after HIV-1infection and eventually starts to mutate in response to error-pronereverse transcription to generate a swarm of closely related variants.The swarm further diversifies in response to selective pressures imposedby both cellular and humoral antiviral immune responses. Virusvariation, driven by the relentless error-prone reverse transcriptionand selection by immune responses, occurs throughout the course of HIVinfection and is perhaps the greatest challenge in the development ofvaccine and therapeutic products. In the present studies we have takenadvantage of mutations occurring early in the course of HIV-1 infectionsto identify specific amino acid substitutions in the HIV-1 envelopeglycoproteins gp120 and gp41 to address the problem of susceptibilityand resistance to neutralization by bNAbs. For this purpose we have madeuse of a large collection of clinical specimens from new HIV infectionscollected in the course of a clinical trial (VAX004) of a candidateHIV-1 vaccine, AIDSVAX. See: Flynn N M, Forthal D N, Harro C D, Judson FN, Mayer K H, Para M F; “Placebo-controlled phase 3 trial of arecombinant glycoprotein 120 vaccine to prevent HIV-1 infection.” TheJournal of infectious diseases 2005; 191:654-65.

This collection of specimens is unique in that they were obtained within6 months of infection (mean 109+/−58 days) from multiple sitesthroughout North America. We reasoned that by studying viruses fromearly infections, sequence variation would be limited compared tosequences collected at later times after infection, and that subsequentmutational analysis would be simpler than that which would be requiredif we used specimens collected from later time points.

Results and Analysis

In initial experiments, we PCR amplified full length envelope genes fromcryopreserved plasma using nested primers of the type described by Li etal. and cloned the swarm of PCR products into a plasmid expressionvector. The vector was specifically designed to permit the constructionof pseudoviruses for use in a well established and validated virusneutralization assay (Monogram Biosciences, Inc—see Schweighardt et al.,2007, J Acquir Immune Defic Syndr 46:1-11 and Whitcomb et al., 2007,Antimicrob Agents Chemother 51:566-75). However, instead of pooling allof the clones together and carrying out neutralization assays with alibrary of cloned genes from each infected individual for neutralizationstudies as had been done previously, we selected 24-48 clones from eachindividual and screened each for infectivity and chemokine receptorusage. We then selected approximately 10 CCR5-dependent pseudotypeviruses with high infectivity for virus neutralization assays. Overall,viruses were prepared from each of 28 individuals and screened forsensitivity and resistance to neutralization (Table 1). In some cases(e.g. subject 108045) all 10 viruses were resistant to neutralization bya panel of four HIV+ sera known to contain broadly neutralizingantibodies (Table 2A). In other cases (e.g. subject 108073) most of theclones were sensitive to neutralization (Table 2B). However inapproximately 85% of the specimens (e.g. subjects 108048 and 108051) wefound a mixture of neutralization sensitive and resistant clones thatshowed differences in sensitivity or resistance to neutralization(Tables 3A and 3B).

After examining the results, 7 clones showing the greatest disparity insensitivity and resistance to neutralization within the same individualwere selected for oligonucleotide sequencing and further analysis. As wehypothesized, sequence variation in several of the sets ofneutralization sensitive and resistant clones was limited and allowedfor the possibility of in vitro mutagenesis to localize the amino acidsresponsible for conferring sensitivity and resistance to neutralizationby HIV+ sera. To explore this possibility, we selected the viruses fromsubject 108060 for further analysis. It can be seen (Table 4A) that 3 ofthe 10 clones analyzed (clones 2, 18, and 24) were relatively sensitiveto neutralization by all 4 HIV+ sera; and of the remaining 7 clones,most were resistant to neutralization by HIV+ sera Z1679, Z1684, andN16) and somewhat sensitive to HIV+ sera from Z23. When the gp160sequences of the neutralization resistant variant (clone 22) and aneutralization sensitive variant (clone 24) were compared (FIG. 1), itwas found that they differed at only seven positions. There were 2 aminoacid differences in gp120, two amino acid differences in the gp41ectodomain, and 3 differences in the cytoplasmic tail of gp41.

To determine which amino acids were responsible for the difference insensitivity to neutralization between clone 22 and clone 24, a series ofmutants were introduced onto the backbone of the neutralizationresistant clone 22 (FIG. 1B). We found (Table 5) that the replacement ofasparagine for serine at position 323 (N323S) in the V3 domain of gp120had no effect on sensitivity to neutralization. Similarly, thesubstitution of asparagine for glycine at position 530 in the C5 domain(N530G) of gp120 had no effect. Replacement of lysine at position 634 ofthe second heptad repeat domain (C34 helix) of gp41 with glutamic acid(K634E) also failed to show a significant difference in neutralizationsensitivity. However the replacement of glutamine for arginine atposition 655 (Q655R) resulted in a remarkable increase in neutralizationsensitivity by all 4 of the HIV+ sera. The difference in neutralizationsensitivity was seen with all four HIV+ sera tested, and titration datafrom the experiments carried out with HIV+ sera Z23 are presented inFIG. 6. This result demonstrated that amino acid substitutions at somelocations in the 34 helix, but not others, can cause a significantchange in sensitivity and/or resistance to neutralization by antibodiesin HIV+ sera.

To understand the impact of this mutation on the structure and functionof the 108060 envelope glycoprotein, we examined the linear and 3dimensional structures of gp41. Examination of the linear structure(FIG. 12) revealed that position 655 was located in the conserved secondheptad repeat of gp41 in a region also known as the C34 helix. This partof the molecule is known to play an integral role in virus fusion andindeed forms an essential component of the 6 coil bundle structure thatis thought to mediate fusion of the viral membrane with T cell membranein the course of HIV infection. Position 655 is also located in the T-20peptide (FIG. 12) that provides the basis for the antiviral drug,FUZEON, that inhibits HIV infectivity by inhibiting virus fusion andentry. Finally the location of this mutation is only eight amino acidsfrom the Membrane Proximal External Region (MPER) of gp41 that is knownto contain two distinct epitopes recognized by the broadly neutralizingmonoclonal antibodies 2F5 and 4E10. Taken together these results suggestthat this mutation occurs in a region that is essential for virus fusionand is in close proximity to a region known to contain other epitopesrecognized by other broadly neutralizing antibodies.

The availability of a 3-D structure of the activated 6 coil structure ofthe gp41 fusion domain allowed us to evaluate the impact of thesubstitution of arginine for glutamine at position 655 on the structureand function of gp41. Using the structure of Chan and Kim we were ableto determine that glutamine at position 655 is located at an internalposition facing the interface with the adjacent between two adjacentgp41 monomers, two turns from the terminus of the C34 helix. Theglutamine 655 side chain contributes two hydrogen bonds that supportboth intra-molecular and inter-molecular interactions. One hydrogen bondis formed by association with glutamine at position 553 of theN-terminal heptad repeat 1 (N36 helix) and the second hydrogen bondinvolves an inter-molecular interaction with the backbone of valine atposition 551 of an adjacent C34 monomer in the 6 coil bundle. Whenviewed in the context of the 6 coiled bundle, the hydrogen bondscontributed by glutamine 655, glutamine 551, and valine 551 form aninter-molecular ring structure that appears to stabilize the 6 coiloligomeric structure. Molecular modeling suggested that replacement ofglutamine with arginine impacts the structure of the 6 coil bundle intwo ways. First the longer arginine side chain disrupts the closepacking of the C34 helix with the N36 helix on the adjacent monomers andprecludes the possibility of a hydrogen bond between the arginine sidechain with valine 551. Although replacement of arginine for glutamine at655 does permit an intra-molecular hydrogen bond between arginine withglutamine 553, this mutation precludes the possibility of theinter-molecular ring of hydrogen bonds that appears to stabilize thequaternary interactions involved in the 6 coil assembly.

Monoclonal Antibody Sensitivity and Envelope Transfer-Sensitivity toneutralization by MAbs and fusion inhibitors. While the structuralanalysis provided insight into the functional consequences of mutationsat position 655, two alternate hypotheses can account for a mechanism bywhich this mutation increases sensitivity to antibody-mediatedneutralization. One possibility is that this mutation is located at ornear an antibody binding site and that the Q655R mutation restores anepitope recognized by a population of neutralizing antibodies present inall four HIV-positive sera. Alternatively, it is possible that thismutation results in a significant conformational change that istransmitted to other parts of gp41 such as the adjacent MPER or thegp120/gp41 trimer complex in such a way as to increase exposure oraccess to antibodies at other locations on the molecule.

To explore these possibilities, antibody neutralization studies werecarried out with a panel of neutralizing MAbs to epitopes in gp120 andgp41 as well as fusion inhibitors targeting either the gp120 or the gp41portion of the HIV envelope glycoprotein. In these studies, we examinedtwo broadly gp41-neutralizing MAbs, 2F5 and 4E10; the broadlyneutralizing b12 antibody able to block CD4 binding to gp120; and 2G12,an antibody that binds to a carbohydrate epitope in gp120. In addition,we tested the antiviral entry inhibitor CD4-IgG, which binds tosequences in gp120 and is able to neutralize lab-adapted CXCR4-dependentclinical isolates at low concentrations (0.01 to 0.1 μg/ml), and primaryclinical isolates of HIV at high concentrations (10 to 100 μg/ml). Wealso examined the sensitivity of envelope mutants to enfuvirtide, apeptide virus entry inhibitor that consists of a gp41-derived peptidethat includes sequences from the C34 helix containing Q655. The resultsof these studies are shown in Table 6, in which the sensitivities ofclone 022 and clone 024 from subject 108060 to neutralizing MAbs werecompared. It can be seen that the neutralization-resistant clone 022 ismoderately sensitive to the 2F5 and 4E10 MAbs specific for the MPER ofgp41 but resistant to neutralization by the b12 and 2G12 MAbs reactivewith gp120. This virus was also sensitive to enfuvirtide and resistantto CD4-IgG. The high CD4-IgG concentration required for theneutralization of this virus is consistent with the concentrationrequired to neutralize other primary, CCR5-dependent viruses. We nextexamined the neutralization-sensitive clone 024 that differs from theneutralization-resistant clone 022 at only seven amino acid positions.We found that this clone was 15- to 20-fold more sensitive to theMPER-specific MAbs (2F5 and 4E10) than the 022 clone. Similarly, theneutralization-sensitive clone 024 was more than 20-fold more sensitiveto CD4-IgG and 3.5-fold more sensitive to neutralization by enfuvirtide(Table 6). Thus, clone 024 exhibited significantly increased sensitivityto neutralization by MAbs and antiviral entry inhibitors as well asantibodies in HIV-positive sera. We then mutated theneutralization-sensitive clone 024 so as to replace R with Q at position655. We found that the resulting mutant (108060_024 R655Q) becameresistant to neutralization and showed a pattern of neutralizationsensitivity closely resembling that of the neutralization-resistantclone 022. Conversely, when we mutated the neutralization-resistantclone 022 to replace Q at position 655 with R, the resulting mutant(108060_022 Q655R), which differed from the parentalneutralization-resistant clone by a single amino acid, exhibited anextraordinary increase in neutralization sensitivity (Table 5). Weobserved a >125-fold increase in sensitivity to CD4-IgG compared to thatof the wild-type clone 022 and a 30- to 35-fold increase in sensitivityto the MPER-reactive antibodies 2F5 and 4E10. We also noted a 17-foldincrease in sensitivity to the antiviral drug enfuvirtide. These resultshighlight the importance of glutamine at position 655 and suggest thatepistatic mutations at other sites in clone 024 moderate sensitivity toneutralization. The results of these studies are remarkable in that theyshow that a single amino acid substitution in gp41 not only conferssensitivity to neutralization by MAbs and entry inhibitors directed togp41 but also increases sensitivity to CD4-IgG, a molecule that binds togp120, an entirely different protein. Thus, the Q655R mutation appearsto cause a conformational change in gp41 that affects not only thebinding of antibodies and entry inhibitors (2F5, 4E10, and enfuvirtide)that bind close to the site of the mutation but also the binding ofanother inhibitor (CD4-IgG) that binds to a site on gp120 located aconsiderable distance from the mutation.

Transfer of the Q655R mutation to related and unrelated viruses. Inorder to determine whether the Q655R mutation could conferneutralization sensitivity and resistance to other viruses, thismutation was introduced into two unrelated viruses highly resistant toneutralization (from subjects 108069 and 108051) that normally possesseda Q at a position corresponding to 655 of the virus from subject 108060(the 108060 virus). The results of these experiments are shown in Table6. Interestingly, we found that the replacement of Q655 with R hadlittle or no effect on neutralization by any of the HIV-positive sera.However, these mutations significantly increased the sensitivity toneutralization by the 2F5 and 4E10 MAbs (25- to 35-fold). Thesemutations also increased the sensitivity to neutralization by the entryinhibitors enfuvirtide and CD4-IgG. Thus, the mutation of Q to R at aposition corresponding to 655 in the 108069 virus increased thesensitivity to enfuvirtide by more than 17-fold and increased thesensitivity to CD4-IgG by more than 20-fold. The 108069 mutant with theQ655R mutation seemed to be somewhat more sensitive to enfuvirtide andpossibly CD4-IgG than the corresponding mutant of the 108051 virus.Together, these results demonstrate that the mutation of Q to R atpositions corresponding to 655 of the 108060 virus confers sensitivityto neutralizing MAbs to the MPER and antiviral compounds targeted to theC34 helix and the MPER of gp41. However, it was interesting that thesemutations failed to increase the sensitivity to bNAbs in HIV-positivesera. We do not know whether neutralizing activity in HIV-positive serais attributable to antibodies binding to the C34 region, the MPER, orother parts of the molecule. It has been recently reported thatantibodies with specificities similar to 2F5 and 4E10 are rare inHIV-positive sera, which might account for the lack of effect.Alternatively, the failure of the Q655R mutation to increaseneutralization sensitivity by HIV-positive sera might be attributable topolymorphisms outside of the MPER and the C34 region that preclude thebinding of otherwise bNAbs. This may well be the case since the 108069and 108051 viruses were selected because of their resistance toneutralization by the HIV-positive sera selected for use in thesestudies.

Expression of envelope proteins derived from the 108060 clone 22 withthe Q655R mutation. In certain embodiments it is desirable to expressthe protein as a fusion protein that includes a non-HIV signal sequenceand a flag epitope for purification. In certain embodiments it isconsidered desirable to delete the furin cleavage site that isresponsible for maturational cleavage of the gp160 precursor into themature gp120 and gp41 proteins.

FIG. 14 shows three pairs of sequences from neutralization sensitive andneutralization resistant viruses. Swam analysis was used to map themutations conferring sensitivity and resistance to broadly neutralizingantibodies in HIV+ sera. Included are sequences from subject 108060 aswell as sequences from subject 108051 and 108048. The preferredsequences for vaccines can be (1) the neutralization sensitive variantenvelope proteins, or (2) the envelope proteins of the resistant viruseswhere a single amino acid substitution (e.g., Q655R) conferringneutralization sensitivity has been created by in vitro mutagenesis, or(3) any sequence derived from of such sequences. This second type ofenvelope protein construct appears to provide very strongimmunogenicity. Insertion of the single amino acid substitution in aneutralization resistant variant envelope protein often results in avirus that is much more sensitive to neutralization than the originalneutralization sensitive variant where there are multiple amino aciddifferences between the neutralization sensitive and resistant variants.An example of this can be seen in Table 5 where clone 22 with the Q655Rmutation is much more sensitive to neutralization than theneutralization sensitive clone 24 variant.

FIG. 14 shows the sequences from subjects 108060, 108051, 108048corresponding to neutralization sensitive and neutralization resistantvariants.

It is interesting to note that the resistant sequence from 108069, whenaltered to include the Q655R substitution, and analyzed usingprotein-blast, identified the following top three most similar sequencealignments:

gb|ABG67916.1| optimized HIV-1 subtype B consensus env gp [syntheticconstruct] Length=850

Score=1482 bits (3836), Expect=0.0, Method: Compositional matrix adjust.

Identities=736/863 (85%), Positives=788/863 (91%), Gaps=22/863 (2%)

gb|AAB64170.1| env polyprotein [HIV-1] Length=854

Score=1461 bits (3783), Expect=0.0, Method: Compositional matrix adjust.

Identities=723/864 (83%), Positives=770/864 (89%), Gaps=20/864 (2%)

gb|ACD41904.1| envelope glycoprotein [HIV 1] Length=855

Score=1459 bits (3777), Expect=0.0, Method: Compositional matrix adjust.

Identities=715/862 (82%), Positives=775/862 (89%), Gaps=15/862 (1%)

Clearly none of these have greater than 85% amino acid identity.

Discussion

In the present studies we describe a novel method useful for mappingepitopes recognized by bNAbs in HIV+ sera as well as mapping mutationsthat confer sensitivity and resistance to virus neutralizing antibodies.The method relies on naturally occurring mutations in the swarm ofclosely related viruses that evolve during the course of HIV infection.Some of these mutations occur at epitopes or contact residues recognizedby broadly neutralizing antibodies, and some of these appear to effect aconformational change that alters the binding of bNAbs at sites that aredistinct from the site of mutation. In previous studies we noted adifference in the binding of a monoclonal antibody between two clones ofthe HIV-1 gp120 envelope protein obtained from a high risk volunteerthat participated in a phase I trial of a candidate HIV vaccine.However, at the time the study was carried out it was not possible tostudy the effect of this mutation in a virus neutralization assaybecause technology was not yet available to re-introduce the mutantenvelope protein back into the virus with assurance that the sequenceswere stable and wouldn't change as a consequence of errors in reversetranscription or selection induced by in vitro culture. However, theadvent of pseudotype virus neutralization assays utilizing HIV envelopegenes incorporated into a stable DNA plasmid vector as opposed toretroviruses with RNA genomes provided the opportunity to take advantageof naturally occurring mutations in HIV envelope genes without the fearof reversion or further mutations. Moreover high throughput sequencingstrategies have since been developed that have allowed us to quickly andconveniently sequence multiple variants from the same individual.

Previous attempts to characterized bNAbs in HIV patient sera have reliedprimarily on immunoadsorbtion studies or on the production of bNAbs fromhuman or mouse B-cells. Immunoadsorbtion studies of HIV+ sera withrecombinant gp120 has shown that some bNAbs appear to recognizeconformation dependent epitopes, some of which are able to block thebinding of gp120 to its cellular receptor, CD4. Studies with monoclonalantibodies prepared from HIV+ individuals have shown that broadlyneutralizing antibodies recognize carbohydrate residues in gp120 (e.g.2G12) or epitopes in the membrane proximal domain of gp41 (e.g. 2F5 or4E10). The best characterized bNAb, 1B12, was isolated from miceimmunized with gp120 and optimized for neutralizing activity by geneticengineering. This antibody binds to a complex conformational epitope andis able to block CD4 binding. However it is not clear whether any ofthese monoclonal antibodies are representative of antibodies found inHIV+ sera, and attempts investigate this possibility remaininconclusive.

In this study we validate the method of using intra-patient variation inthe HIV envelope protein in the context of a pseudotype virusneutralization assay to identify mutations that sensitivity andresistance of viruses to neutralization by broadly neutralizingantibodies. Using this method we expect to be able to identify specificepitopes recognized by bNAbs as well as amino acid mutations that alterthe sensitivity and resistance of viruses to neutralization byantibodies. In the present studies we have identified a single aminoacid substitution (Q655R) in the C34 helix of gp41 that appears to playan important and previously unrecognized role in maintaining theintegrity of the 6 coil bundle in the viral membrane fusion apparatus ofHIV-1. X-ray crystallography studies demonstrate that this residuecontributes two hydrogen bonds: one mediating an intra-molecularinteraction with the N36 helix on the same monomer and the othermediating an inter-molecular interaction with the N36 helix on anadjacent monomer. This mutation appears to affect sensitivity toneutralization by bNAbs by altering 4 distinct interactions. First theQ655R mutation breaks a hydrogen bond that mediates an intra-molecularinteraction (Q at position 655 of the C34 helix with valine at position551 of the N36 helix). Second, the Q655R mutation disrupts aninter-molecular interaction (Q at position 655 with valine at position553 in the N36 helix) with an adjacent monomer. Third, the longerarginine side chain in the Q655R mutation appears to alter theinter-helix packing interface between adjacent monomers by stericallyhindering the close association between the C34 helix and the N36 helixon adjacent monomers. Finally, the Q655R mutation appears to prevent theformation of a ring structure involving 12 hydrogen bonds in the 6 coilbundle that occurs upon formation of the gp41 fusion complex. Althoughit is possible that R655 is able to form an intra-molecular hydrogenbond with position 551, it does not appear likely that this mutationallows for replacement of the inter-molecular hydrogen bond with aresidue on the adjacent N36 helix essential for the formation of aninter-molecular hydrogen bonded ring structure.

The location and structural impact of the 655 mutation described in thispaper appears to be fundamentally different from another recentlydescribed gp41 variant that that affects sensitivity and resistance toneutralization by bNAbs. First, the neutralization sensitive phenotypein this study requires two mutations: an isoleucine to valinesubstitution at position 675 (I675V) in the MPER and a threonine foralanine substitution at position 569 (T569A) in the first heptad repeatdomain (N36 helix) of gp41. The MPER is a well known target of virusneutralizing monoclonal antibodies and is structurally distinct from theC34 helix. The T569A mutation does appear to occur at the interface ofthe intra-molecular interaction between the N36 and C34 helices. In thiscase, the substitution of the longer threonine for alanine at position569 appears to preclude a classical “knob in hole” interaction betweenadjacent helices and does not appear to affect inter-molecularinteractions.

Since the 6 helix coil structure appears to be a conserved structuralelement fundamental to many biologic processes involving membranefusion, it may well be the case that hydrogen bond ring structures ofthe type we have identified for HIV-1 are present and essential formaintaining the functional integrity of coiled-coil bundles required formembrane fusion in other viruses such as influenza, Moloney leukemiavirus, Ebola virus, and Visna virus.

If hydrogen bonded ring structures of the type we have identified forHIV are found to be present in other coiled-coil bundles involved inmembrane fusion, they may provide a novel rationale for the developmentof vaccines for the prevention and treatment of other virus infections.Many viruses are thought to use homologous 6 coil bundles to mediatemembrane fusion and virus entry, see: Flint S J, Enquist L W, Krug R M,Racaniello V R, Skalka A M. Principles of Virology. 2nd ed.: ASM Press;2004. We would expect that viruses with similar mutations that affecthydrogen bonded ring structures that stabilize 6 coil bundles may alterthe structure of the virus in such a way as to expose importantneutralizing sites and facilitate recognition by the immune system. Wesuggest that HIV envelope glycoproteins with mutations in gp41 thatdestabilize the 6 coil bundle structure such as that seen in clone 24from subject 108060 may prove to be superior vaccine immunogens byproviding better exposure of epitopes to B-cell receptors or T-cellsrequired for the formation of broadly neutralizing antibody responses.

Detailed Description of the Invention (SC2010-117) Relating to Method toImprove the Immunogenicity of Vaccine Antigens by Modification ofCleavage Sites in HIV-1 gp120

A major goal in HIV vaccine research is the identification of vaccineimmunogens able to elicit broadly neutralizing antibodies (bNAbs) andprotective cellular immune responses. After more than 25 years ofresearch, antigens with these properties have yet to be described.However, several studies have demonstrated that recombinant envelopeglycoproteins are able to adsorb broadly neutralizing antibodies fromHIV+ sera. Thus the epitopes recognized by bNAbs are present onrecombinant proteins, but they are not immunogenic. These results raisedthe possibility that alteration of the pattern of antigen processingmight refocus the immune response to regions of the envelopeglycoprotein that are better able to elicit protective immunity.

The inventors have discovered various protease cleavage sites on HIVgp120 recognized by three major human proteases (cathepsins L, S, and D)important for antigen processing and presentation. Remarkably, six ofthe eight sites identified were highly conserved and clustered inregions of the molecule associated with receptor binding and/or thebinding of neutralizing antibodies. These results suggested that HIV mayhave evolved a novel mechanism of immune escape by taking advantage ofantigen processing enzymes in order to insure that epitopes recognizedby neutralizing antibodies are labile and destroyed by proteolysisbefore they can stimulate protective immune responses. The results fromthese suggest the possibility that HIV regulates the immunodominance ofMHC class II restricted immune responses by limiting the number andlocation of protease cleavage sites.

The invention encompasses improved vaccine antigens that may be producedby mutation of conserved protease cleavage sites in various viralenvelope glycoproteins. The invention details a method of improving theimmunogenicity of vaccine antigens by preserving the structure ofepitopes recognized by virus neutralizing antibodies that are otherwiseinactivated in vivo by exposure to cell associated or secretedproteases.

The method entails: 1) determination of the location of proteasecleavage sites on virus envelope proteins by in vitro analysis wherepurified envelope proteins are treated with serum or cellular proteasesin vitro, and determination of the identity/location of the proteasecleavage sites by standard techniques such as Edmund sequencedegradation or mass spectroscopy. 2) Bioinformatic analysis to align thesequences of one virus envelope protein with envelop proteins fromdifferent strains of the same virus to determine which protease cleavagesites are conserved and to determine which cleavage sites are located atpreviously described neutralizing epitopes or receptor binding sites. 3)In vitro mutagenesis to inactivate conserved protease cleavage sites insuch a way as to preserve the binding of neutralizing antibodies and/orreceptor binding. 4) Screening mutagenized envelope proteins forimproved immunogenicity relative to the wild type virus protein bycomparing the neutralizing activity of experimental antisera produced insmall animal (e.g. rabbits, rat, mice, guinea pigs) immunogenicitystudies.

Certain embodiments of the invention include:

A virus envelope protein [such as gp120 from the MN strain of HIV whereconserved protease cleavage sites in regions important for receptorbinding or the binding of neutralizing antibodies sites are mutated byamino acid replacement to prevent protease cleavage while at the sametime preserving the antigenic structure of the molecule as defined bythe ability to stimulate the formation of neutralizing antibodies (whenused as an immunogen) or be recognized by neutralizing antibodies (whenused as an antigen).

A virus envelope protein where protease cleavage sites recognized byserum or cellular proteases are deleted or inactivated or otherwiseprotected from protease cleavage by in vitro mutagenesis.

A virus envelope protein used as a vaccine antigen where in vitromutagenesis of conserved cleavage sites protects the neutralizingepitopes from proteolytic degradation in an in vivo environment.

A virus envelope protein where conserved protease cleavage sites locatedwithin epitopes recognized by neutralizing antibodies are deleted orinactivated or otherwise protected from protease cleavage by in vitromutagenesis in such a way as to preserve the ability of the epitope tobind specifically to neutralizing antibodies.

A virus envelope protein described above where the protease cleavagesites are specific for the antigen processing enzymes cathepsin L,cathepsin S, or cathepsin D.

A virus envelope protein described above where the protease cleavagesites are specific for the serum protease thrombin, or the cellassociated protease, tryptase, or the inflammation associated proteasessuch as elastase.

A virus envelope protein described above where the protease cleavagesites are specific for other members of the cathepsin family such ascathepsin B, K, N.

A virus envelope protein as described above where the protein consistsof monomeric or oligomeric fragments of the HIV envelope protein gp160such as gp120, gp140, or gp41.

A virus envelope protein as described above where the protein consistsof monomeric or oligomeric fragments of the influenza virushaemagglutinin (HA1/HA2) any strain of influenza (e.g. H1N1).

A virus envelope protein as described above where the protein consistsof monomeric or oligomeric fragments of glycoprotein D from HerpesSimplex virus type 1 or type 2.

A virus envelope protein as described above where the protein consistsof monomeric or oligomeric fragments of any virus envelope protein forcellular receptor binding.

A virus envelope protein wherein one or more conserved cleavage sitesare protected from protease cleavage by in vitro mutagenesis, andwherein said cleavage sites are selected from the cathepsin cleavagesites on MN-rgp120 shown in Table 1.

Other embodiments include the following:

1. A virus envelope protein wherein one or more conserved cleavage sitesare protected from protease cleavage by in vitro mutagenesis.

2. The virus envelope protein of claim 1 wherein said cleavage sites areselected from the cathepsin cleavage sites of MN-rgp120 as shown inTable 1, and homologues thereof.

3. The virus envelope protein of claim 1 wherein said cleavage sites theprotected from protease cleavage by deletion, mutation, chemicalmodification (e.g. methylation, acetylation, glycosylation, etc).

4. The virus envelope protein of claim 1 formulated with an excipient,carrier or adjuvant for use as a vaccine.

5. A vaccine formulation comprising an HIV envelope glycoprotein and aprotease inhibitor.

6. The vaccine formulation of claim 5 wherein the protease inhibitor isa cathepsin.

7. The vaccine formulation of claim 6 wherein the cathepsin is humancathepsin L, S or D.

8. A vaccine formulation comprising an HIV envelope glycoprotein whereinone or more conserved cleavage sites of the HIV envelope glycoprotein isprotected from protease cleavage by in vitro mutagenesis, and whereinthe one or more conserved cleavage sites is selected from the cathepsincleavage sites of MN-rgp120 as shown in Table 1, and homologues thereof.9. The vaccine formulation of claim 8 wherein the cleavage sites areprotected from protease cleavage by deletion, mutation, methylation oracetylation.10. A method for treatment or prevention of a viral infection, themethod comprising administering to a subject the vaccine formulation ofclaim 5.11. A method for treatment or prevention of a viral infection, themethod comprising administering to a subject the vaccine formulation ofclaim 8.12. A method for treatment or prevention of a viral infection, themethod comprising contemporaneously administering to a subject acomposition comprising an HIV envelope glycoprotein and a proteaseinhibitor.13. The method of claim 12 wherein the protease inhibitor is aninhibitor of a cathepsin.

Additional embodiments include the following: 1. A virus envelopeprotein where conserved protease cleavage sites serve to inactivateepitopes recognized by neutralizing antibodies and are responsible forthe lack of protective immune responses when used as a vaccine antigen.2. A virus envelope protein where conserved cleavage sites recognized byserum or cellular proteases are deleted or inactivated by in vitromutagenesis. 3. A virus envelope protein used as a vaccine antigen wherein vitro mutagenesis of conserved cleavage sites protects theneutralizing epitopes from proteolytic degradation after parenteralinjection 4. A virus envelope protein where conserved protease cleavagesites located within epitopes recognized by neutralizing antibodies aredeleted or inactivated by in vitro mutagenesis in such a way as topreserve the ability to bind neutralizing antibodies. 5. A virusenvelope protein described in claim 2 where the protease cleavage sitesare specific for the antigen processing enzymes: cathepsin L, cathepsinS, or cathepsin D. 6. A virus envelope protein described in claim 2where the protease cleavage sites are specific for the serum proteasethrombin, or the cell associated protease, tryptase, or the inflammationassociated proteases such as elastase. 7. A virus envelope proteindescribed in claim 2 where the protease cleavage sites are specific forother members of the cathepsin family such as cathepsin B, K, N. 8. Avirus envelope protein as described in claim 2 where the proteinconsists of monomeric or oligomeric fragments of the HIV envelopeprotein gp160 such as gp120, gp140, or gp41. 9. A virus envelope proteinas described in claim 2 where the protein consists of monomeric oroligomeric fragments of the influenza virus haemagglutinin (HA1/HA2) ofany strain of influenza (e.g. H1N1). 10. A virus envelope protein asdescribed in claim 2 where the protein consists of monomeric oroligomeric fragments of glycoprotein D from Herpes Simplex virus type 1or type 2. 11. A virus envelope protein as described in claim 2 wherethe protein consists of monomeric or oligomeric fragments of any virusenvelope protein for cellular receptor binding.

After many years of research, vaccine immunogens able to elicitprotective immunity in humans have yet to be described. Although it hasbeen possible to produce recombinant proteins that accurately replicatethe complex structure of HIV envelope glycoproteins, these antigens havenot been able to elicit broadly neutralizing antibodies or protectiveimmune responses. The fact that recombinant proteins can bind with highaffinity to the CD4 and chemokine receptors used by HIV-1 for attachmentand entry, and can adsorb virus broadly neutralizing antibodies fromHIV-1+ sera suggests that while the neutralizing epitopes of recombinantimmunogens possess the proper antigenic structures, these structures areimmuno-recessive and simply not immunogenic. Over the last decade,several different approaches have been employed in order to createimmunogens able to elicit broadly neutralizing antibodies. Thesestrategies have included 1) efforts to duplicate and/or stabilize theoligomeric structure of HIV envelope proteins 2) the creation of minimalantigenic structures lacking epitopes that conceal importantneutralizing sites, and 3) prime/boost strategies combining proteinimmunization with DNA immunization or infection with recombinant virusesin order to stimulate the endogenous synthesis and presentation of HIVimmunogens (13, 26, 27, 69). However, none of these approaches hasresulted in a clinically significant improvement in antiviral immunityor HIV vaccine efficacy. Efforts to elicit protective cellular immuneresponses (e.g. cytotoxic lymphocytes) using recombinant virus vaccineshave likewise been disappointing. In fact such vaccines may havepromoted HIV infection rather than inhibiting it.

The inventors describe a new approach to re-engineering theimmunogenicity of HIV envelope proteins in order to improve the potencyand specificity of humoral and cellular immune responses. The approachis based on defining the sites recognized by proteases important forantigen processing as well as other plasma and cell associatedproteases. The inventors reasoned that mutation of the sites recognizedby proteases essential for antigen processing and presentation mightincrease the level of helper T cells and refocus the specificity of theantiviral immune response to favor the development of protectiveimmunity. Both humoral and cellular immune responses depend onproteolytic degradation in connection with antigen processing andpresentation mediated by professional antigen presenting cells(macrophages, dendritic cells, and B-cells). Normally, proteins ofintracellular origin are processed by the proteosome, a 14-17 subunitprotein complex located in the cytosol. Proteins of extracellular originare processed in lysosomes or late endosomes of antigen presenting cells(APCs). The resulting peptide epitopes are then loaded into MHC class Imolecules and presented to CD8 or CD4 T-cells on the surface of APC.Within the endosomes and lysosomes of APCs, there are cathepsins, acidthiol reductase, and aspartyl endopeptidase. The enzymes perform twoactivities: degrading endocytosed protein antigens to liberate peptidesfor MHC Class II binding, and removal of the invariant chain chaperone(4). Although all cathepsins can liberate epitopes from a diverse rangeof antigens (14), only cathepsins S and L have nonredundant roles inantigen processing in vivo (reviewed in Hsing and Rudensky 2005).Cathepsin L is expressed in thymic cortical epithelial cells but not inB cells or dendritic cells, while the distribution of cathepsin S is inboth types of antigen presenting cells. Unlike cathepsins L and S, whichare cysteine proteases and active at neutral pH, cathepsin D is anaspartic protease, active at acidic pH, and participates in proteolysisand antigen presentation in connection with MHC class I and class IIantigen presentation pathways established for CD4 and CD8 T-cells. Whenconsidering the use of envelope proteins as potential vaccines, theroute of immunization, formulation (e.g. adjuvants), protein folding,disulfide bonding, and glycosylation pattern all determine whichpeptides are available for MHC restricted presentation.

Previous studies provided evidence that cathepsins B, D, and L areinvolved in antigen processing of gp120, but the specific cleavage siteswere not defined (Chien, P. C. 2004. Human immunodeficiency virus type 1evades T-helper responses by exploiting antibodies that suppress antigenprocessing. J Virol 78:7645-52.). In the present studies, we: 1)describe the location of eight protease cleavage sites on HIV-1 gp120recognized by cathepsins L, S, and D involved in antigen processing, 2)determine the extent to which they are conserved, and 3) evaluate theeffect of cathepsin cleavage on the binding of gp120 to CD4-IgG andneutralizing antibodies. The results obtained provide new insights intothe basis of envelope immunogenicity that may prove useful in thedevelopment of HIV vaccine antigens.

Materials and Methods.

Proteins, Enzymes and Enzyme Inhibitors.

Recombinant gp120 from the MN strain of HIV-1 (MN-rgp120) was producedin Chinese hamster ovary (CHO) cells by Genentech, Inc. (South SanFrancisco, Calif.). MN-rgp120 was a major component of the candidate HIVvaccine, AIDSVAX, B/B (29). Purified human cathepsins L, S and D as wellas the cathepsin L and D inhibitors N-Acetyl-Leu-Leu-Methional calpainInhibitor II (ALLM) and Pepstatin A were obtained from Biomol(Philadelphia, Pa.).

Monoclonal Antibodies.

The broadly neutralizing, CD4 blocking monoclonal antibody (MAb) b12(10, 57) was obtained from Polymun (Vienna, Aus). The virus entryinhibitor, CD4-IgG (11) and MAbs able to neutralize the MN strain of HIVreactive with the V3 domain (1026), and the C4 domain (13H8) wereobtained from Genentech, Inc. (S. San Francisco, Calif.) and have beendescribed previously (54, 55). Polyclonal antibody D7324 was purchasedfrom Aalto Bio Reagents Ltd. (Dublin, Ireland). HRPlabeled goatanti-human IgG and goat anti-mouse IgG+M were obtained from AmericanQualex Antibodies (San Diego, Calif.).

Cathepsin Digestions for Cleavage Site Analysis.

Fifty μg of MN-rgp120 in 25 μl of 100 mM sodium acetate, pH 5.5digestion buffer was mixed with 0.5 μg cathepsin L (protease to proteinratio 1:100). The reaction was performed at 37° C. Aliquots of 3 μl weretaken at 15 min, 30 min, 60 min, 120 min, 240 min, 420 min and thedigestion was stopped by rapid cooling in liquid nitrogen. An additional3 μl aliquot was taken after overnight incubation at room temperature.The aliquots of cathepsin L digestion were mixed with 3 μl of 3×reducing polyacrylamide gel electrophoresis (PAGE) sample buffer (5%SDS, 5% β-mercaptoethanol, 40% glycerol and 200 mM Tris, pH 6.8) andboiled for 2 min. The collected samples were run in two 4-12% Bis-Trispre-cast gels (Invitrogen, Carlsbad, Calif.). Digested fragments werevisualized either by direct Coomassie blue staining or on immunoblotsafter transferred to a PVDF membrane (Millipore Immobilon PSQ). Forsequencing peptides on PVDF membranes, bands were cut out andtransferred to the Molecular Structure Facility at the University ofCalifornia, Davis for N-terminal protein sequencing by Edmandegradation. The same experimental procedure was carried out forcathepsin S and D digestion except for the digestion buffer, which was50 mM sodium phosphate, pH 6.5, with 50 mM sodium chloride for cathepsinS, and 100 mM sodium acetate, pH 3.3 for cathepsin D.

Cathepsin Digestions for ELISA Experiments.

To prepare cathepsin L digested MN-rgp120 for ELISA experiments, 25 gMN-rgp120 in 50 μl of 100 mM sodium acetate, pH 5.5 digestion buffer wasmixed with 1 μg cathepsin L (protease to protein ratio 1:25) at 37° C.for overnight incubation and followed by 1 μl ALLM (25 mg/mL in DMSO)solution to stop the digestion reaction. For cathepsin D treatedMN-rgp120, 25 μg of MN-rgp120 was mixed with 1 μg of cathepsin D in 50μL buffer (100 mM sodium acetate, pH 3.3) at 37° C. for 1 h. Pepstatin A(11 g at 25 mg/mL in DMSO) solution was added to stop cathepsin Dactivity.

ELISA of Monoclonal Antibodies and CD4-IgG Binding to Cathepsin DigestedMN-Rgp120.

Wells of microtiter plates (Immunosorb II, Becton-Dickenson, MountainView, Calif.) were coated with 100 μL of the polyclonal antibody D7324solution (2 μg/mL in PBS buffer) overnight at 4° C. The wells wereblocked with 200 μL of blocking buffer (1% BSA in PBS) and incubated at37° C. for 1 h. After rinsing with washing buffer (0.05% Tween 20 inPBS), 100 μL of cathepsin L treated, cathepsin D treated or undigestedMN-rgp120 solution was added to each well (2 μg/mL in blocking buffer)and incubated for 1 h at 37° C. Then, after washing, MAbs were added andfive-fold serial dilutions were carried out, starting with 25 ug/mL ofMAb b12 and 5 μg/mL of all of the other MAbs, and incubated for 1 h at37° C. After washing three times with blocking buffer, 100 μL of HRPlabeled goat anti-human IgG or goat anti-mouse IgG+M solution (1:10000in blocking buffer) was added and incubated for 1 h at 37° C. Finally,after washing, 100 μL of 0.4 mg/mL o-phenylenediamine dihydrochloride(Sigma Aldrich Chemicals, St. Louis Mo.) solution was added andincubated at room temperature for 10 min, followed by 100 μL of 3Msulfuric acid to stop the reaction. The O.D. was measured by SpectraMax190 (Molecular Devices) at 490 nm.

Prediction of Cleavage Sites by Computational Methods.

Envelope glycoprotein sequences were obtained from the Los Alamos HIVsequence database and aligned using MAFFT. The sequence for gp120 fromthe MN strain of HIV-1 used in these studies, MNGNE, differs from thesequence of Gurgo et al. and has been published previously. To determinethe location of predicted cathepsin cleavage sites in MNrgp120, we usedthe PoPs program developed by Boyd et al., and cleavage specificityalgorithms for cathepsins L, S and B generated by Choe and the cathepsinD recognition sequence of Dunn et al.

Conservation Study of Identified Cleavage Sites.

Three datasets were used to investigate the sequence conservation ofcathepsin cleavage sites. The VAX004 dataset was obtained from the GSIDHIV data browser (“http:” followed by “//www.gsid” followed by “.org”),which includes 1047 clade B envelope glycoprotein sequences from 349individuals with recent HIV infections. A dataset of acute and recentclade B infections containing 2908 envelope glycoprotein sequences from102 infected individuals was obtained from the studies of Keele et al.Finally a listing of clade specific reference sequences as well as adataset containing 1766 envelope glycoprotein sequences from isolatescollected world-wide at various undefined times after HIV infection wasobtained from the Los Alamos HIV Sequence Database (“http:” followed by“//www.hiv.lanl” followed by “.gov/”). The sequences from all threedatabases were aligned using MAFFT.

Results.

Computational Methods to Locate Protease Cleavage Sites.

Cathepsins L, S, and D are known to play an important role in antigenprocessing and presentation. In initial studies we used computationalmethods (see Materials and Methods) to determine whether gp120 waslikely to possess cleavage sites recognized by cathepsins known to beimportant for antigen processing. For these studies we examinedsequences with the prediction algorithm (6) set for maximum stringency.The results of these studies (FIG. 13) suggested that MN-rgp120 waslikely to possess multiple cathepsin cleavage sites. However, becausecathepsin cleavage sites are difficult to predict, and limitedinformation is available (17); MEROPS Peptidase Database (“www.merops.”followed by “sanger.ac” followed by “.uk”), we reasoned that actualprotease digestion studies would be required to reliably identify thenumber and location of these sites.

Mapping Cathepsin L Cleavage Sites.

Initially we examined sensitivity of MNrgp120 to digestion by cathepsinL. A time-course experiment is shown in FIG. 7, Panel A. We found thatcathepsin L digestion resulted in six proteolytic fragments. Because oftheir size, these fragments could not be analyzed by mass spectrometrybut rather required analysis by Edman sequence degradation. The size,location, and experimentally determined N-terminal sequence of thepeptides isolated is shown in Table 1. The cleavage site shown in thistable represents the P1 and P1′ residues located on either side of thecleavage site according to standard protease substrate nomenclature. Alisting of flanking residues ranging from P4 to P4′ is provided insupplemental table S1. We found that digestion with cathepsin L resultedin a 70 kD fragment and a 50 kD fragment appeared within fifteen minutesof treatment. Edman degradation showed the first five amino acids in theN-terminal of the 50 kD fragment are GTIRQ, which revealed that thecleavage site is located between the K327-G328 bond in the V3 domain.The N terminus of the 70 kD fragment is derived from cleavage betweenthe A28-L29 bond in the glycoprotein D flag epitope at the N-terminus ofMN-rgp120, resulting in the L29-A30-N31 N-terminal sequence. Thekinetics of the appearance of these two fragments indicates theMN-rgp120 was first attacked at the V3 domain cleavage site K327-G328resulting in two fragments, the 70 kD fragment and the 50 kD fragment.The 50 kD fragment was subsequently degraded at longer digestion timesto yield additional fragments (FIG. 7, Panel A and 8). The Edmandegradation confirmed that the resulting 45 kD and the 35 kD fragmentsare originally from the 50 kD fragment because both include the sameN-terminal sequence GTIRQ as the 50 kD fragment. Although the C-terminalsequences of the 50 kD, 45 kD and 35 kD fragments are not known, atleast two cathepsin L cleavage sites in the 50 kD fragment areindicated, which result in the 45 kD and 35 kD fragments. The N-terminalamino acid sequence of the resulting 20 kD fragment and the 14 kDfragment proved that there are two cathepsin cleavage sites within theC4 domain. The first four amino acids of the N-terminal of the 20 kDfragment and the 14 kD fragment are KAMY and APPI respectively. Thus,two cathepsin L cleavage sites located between the G431-K432 andY435-A436 bonds were identified. However, because the molecular weightdifference between 20 kD and 14 kD is about 6 kD while the N-terminalsequence difference between the 20 kD and 14 kD is only four aminoacids, we deduced that another cathepsin L cleavage site must be presentwithin the C-terminus of MN-rgp120.

Mapping Cathepsin S Cleavage Sites.

We next examined the ability of cathepsin S to digest gp120 using thesame methods. The result of a time-course experiment is shown in FIG. 7,Panel B. It can be seen that six degradation products were visible onSDSPAGE gels. The size of the peptides isolated, the N-terminal sequenceand the location within gp120 is shown in Table 1. Compared to thecathepsin L digestion, the kinetics of the cathepsin S digestion weremuch more rapid and indicated significantly increased sensitivity tocathepsin S. Six major digestion fragments appeared on the SDS-PAGE gelwithin fifteen minutes of cathepsin S digestion, indicating greaterexposure or accessibility of cathepsin S cleavage sites compared tocathepsin L. Because of the rapid digestion by cathepsin S, it wasn'tpossible to determine whether there was a kinetically distinct, ordereddegradation of gp120 as seems to be the case with cathepsin L. Rather,cathepsin S appears to follow a different digestion pathway where theprotease generates multiple fragments in a very short time frame.Analysis of five cathepsin S digest fragments (e.g. 60 kD, 50 kD, 38 kD,18 kD and 12 kD) identified four distinct cathepsin S cleavage sites(FIG. 8). Two of these were located in the C2 domain and occurredbetween Q208-A209 (60 kD) and S261-T262 (50 kD). The third cathepsin Scleavage site occurred in the V3 domain and involved the bond joiningT322-T323 (38 kD). Finally an additional cleavage site was located inthe C4 domain and occurred between Y435-A436 (18 kD and 12 kD) which isalso a cathepsin L cleavage site. Fragments located N-terminal to the C2domain were not recovered, suggesting that this region of the moleculecontains multiple yet undefined cathepsin S cleavage sites. It ispossible that some of these yield 3.5 kD fragments, since the final 3.5kD band on the PAGE gels appeared to be heterogeneous, with multiplefragments migrating at the same position.

Mapping Cathepsin D Cleavage Sites.

A complicated digestion pattern was observed in the digestion ofMN-rgp120 with cathepsin D (FIG. 7, Panel C). Eleven digestion fragmentswere visualized on the SDSPAGE gel, but only eight fragments were ableto be characterized by Edman degradation due to heterogeneity in bandsand/or low abundance. Four fragments (55 kD, 52 kD, 30 kD and 12 kD)share a common N-terminal sequence VVIRS (SEQ ID NO: 14), which islocated in the C2 domain, suggesting a cleavage site E274-V275. Based ondifferences in molecular weights, we have deduced that additionalcathepsin D cleavage sites occur in the V3, C3, V4 and C4 domains (FIG.8). The N-terminal sequencing of the 20 kD and 70 kD fragment indicatedanother cathepsin D.

Cleavage site in the V2 domain at the bond between residues L181-Y182. Athird cathepsin D cleavage site Gly25-Lys26 occurred close to theN-terminus and produced a 4 kD and a 5 kD fragment. The location ofcleavage sites relative to conserved and variable domains as well asdisulfide bonds was mapped onto the 2-dimensional structure of Leonardet al. (49) and is shown in FIG. 9. In total, nine cathepsin cleavagesites were identified, one in the N-terminal flag sequence, one in theV2 domain, three in the C2 domain, two in the V3 domain and two in theC4 domain.

Localization of Cathepsin Cleavage Sites on the 3-Dimensional Structureof gp120.

The cathepsin L, S and D cleavage sites identified in these experimentswere mapped onto the 3-dimensional structure gp120 (FIG. 10) of Huang etal. (41). It was clear from these studies that the cathepsin cleavagesites are not randomly distributed throughout the 3-dimensionalstructure. Remarkably, they appeared to cluster in regions of functionalsignificance, often in close proximity to the binding sites for the CD4and chemokine co-receptors and/or epitopes recognized by neutralizingantibodies (Table 1). For example, the cathepsin S cleavage sites in theC2 (Q208-A209) and the C4 domains (Y435-A436) and the cathepsin L sitesat G431-K432 and Y435-A436 are located in close proximity in the 3dimensional structure of gp120. The K432 residue and the Y435 residuesare known to be contact residues for chemokine receptor binding, and theQ208 residue is one amino acid away from K207 that is also known to be achemokine receptor contact residue (22, 64, 65). Additionally, the G431residue is located 2 amino acids away from a string of six amino acidresidues 424-429 known to be contact residues for CD4 binding (46). V429which is at the P2 position for the cathepsin L recognition site isknown to be a contact residue for both CD4 and the broadly neutralizingb12 MAb (88). Finally the Q208-A209 cathepsin S cleavage site is 3 aminoacids away from K212 also known to be a CD4 contact residue. Twoadditional cathepsin cleavage sites occur in the C2 domain. Of these,position T262 in the S261-T262 cathepsin L cleavage site is known to bea contact residue for the broadly neutralizing b12 MAb (88); whereas thecathepsin D cleavage site (E274-V275) was the only cathepsin cleavagesite that was not part of, or adjacent to, a receptor or neutralizingantibody binding site. Two cathepsin cleavage sites were identified inthe V3 domain. The V3 domain is known to be an important determinant ofchemokine receptor usage (18, 85) and is known to possess epitopesrecognized by a variety of neutralizing antibodies. The cathepsin S site(T322-T323) is located one amino acid away from the crown of the V3 loopcontaining the GPGRAF (SEQ ID NO:40) sequence important for the bindingof multiple neutralizing antibodies (21, 54, 67). The cathepsin Lcleavage site at K327-G328 is four amino acids from the cathepsin S sitebetween the stem and the crown of the V3 loop. Finally, a singlecathepsin D site involving residues L181-Y182 was located in the V2domain. The V2 domain is known to possess multiple epitopes forneutralizing antibodies (52, 59) and contains the newly describedreceptor binding site for the alpha-4-beta-7 integrin (2).Interestingly, position L181-Y182 cleavage site is located one aminoacid away from the LDI/LDV recognition sequence required foralpha-4-beta-7 binding to gp120.

Conservation of Cathepsin Cleavage Sites.

An important question in these studies was to determine which if any ofthe cathepsin protease sites was conserved. A conserved pattern ofcathepsin cleavage sites would suggest conservation of the MHC class IIrestricted immune response. In view of the high degree of sequencevariation within the HIV virus, and the fact that the envelope proteinis the most variable of all of the HIV proteins, it was uncertainwhether any of the sites would be conserved. In initial studies, wealigned the MN-rgp120 and HXB2 gp120 sequences with twelve referencesequences: two from each of four major group clades: A, C, D, E (crfA/E), plus two from the chimpanzee isolate, HIVcpz, and two simianimmunodeficiency virus (SIV) sequences (SIVMac 251 and SIVMac239). Theresults of this analysis are shown in Table 2 where both the locationand conservation of the sites recognized by cathepsins L, S, and D canbe seen along with the locations of predicted cathepsin cleavage sites.This analysis of the residues occurring at the P1 and P1′ positionsshowed a high level of conservation at 6 of the 8 cathepsin cleavagesites.

Remarkably, two sites, including the cathepsin S sites S261-T262 site inthe C2 domain and the cathepsin L site at position G431-K432 in the C4domain, were conserved in the reference strains of the major group HIVclades, the HIV cpz strains, and the SIV strains. A high level ofconservation (˜98%) was also noted at the Q208-A209 cleavage site in theC2 domain, and the Y435-A436 site in the C4 domain. A somewhat lower(81-92%) level of conservation was also noted at the L181-Y182 site inthe V2 domain; however, in this case the MN strain is unusual in that Lreplaces F at position 181. The highly conserved nature of these sitessuggests that they are important for virus function or survival and havebeen preserved by positive selection across species and time.

To further explore the conservation of cathepsin cleavage sites, weexamined the three independent HIV sequence datasets. One dataset (GSIDHIV Sequence Database) included 1047 gp120 sequences from 349individuals with new and recent HIV infections (less than 6 months postinfection) from different cities throughout North America (29). A seconddataset was obtained from the studies of Keele et al. (44) consisting of2908 sequences from 102 new and acute infections collected in the UnitedStates. The third HIV dataset examined was the Los Alamos HIV Sequencedatabase, comprising 1766 gp120 sequences collected from world-wideisolates that included sequences from the 1980s through the presenttime. Most of these sequences were from chronic HIV infections. Theresults of this analysis are presented in Table 2.

We found a very high level of conservation (i.e. >96%) in the Q208-A209and S261-T262 cathepsin cleavage sites in the C2 domain, and theG431-K432 and Y435-A436 cleavage sites in the C4 domain of gp120. In thecase of the 431-432 cleavage site, a significant discrepancy was notedbetween the Los Alamos dataset and the VAX004 and Keele datasets.Further analysis indicated that this result could be attributed toclade-specific polymorphism, where clade B viruses typically possessed Kat position 432, while other clades typically possessed R at thisposition. The F181-Y182 cleavage site in the V2 domain was also highlyconserved (i.e., >80%); however the sequence of HIVMN was unusual inthat K replaced F at position 181.

Effect of Cathepsin Cleavage on the Binding of CD4-IgG and NeutralizingAntibodies.

Based on the location of cathepsin cleavage sites at or near receptorbinding sites and epitopes recognized by neutralizing antibodies, it wasof interest to determine whether cathepsin cleavage actually affectedthe binding of antibodies to these sites. The binding of monoclonalantibodies to cathepsin treated and untreated MN-rgp120 was investigatedby ELISA (FIG. 11). One concern in performing this assay was thepossibility that enzyme cleavage would release small peptide fragmentsthat would not be captured onto the microtiter plate. Examination of theproteases cleavage sites in relation to the disulfide structure showedthat proteolysis of the peptide backbone would not necessarily releasemultiple peptide fragments since most would remain associated by virtueof disulfide bonds. Thus, treatment with cathepsin L should only releasea small 4 amino acid peptide, K432-Y435, from the C4 domain. Treatmentwith cathepsin D might split the molecule into two large fragments byvirtue of the cleavage site located at position 274 in the C2 domain andmight also result in the release of an undefined 4-5 kD fragment fromthe C1 domain. Treatment with cathepsin S should have the largest effectand should result in the loss of the C1, V1, V2, and C2 domains. Forthis reason, we studied antibody binding to only cathepsin L andcathepsin D treated molecules. The panel of MAbs used for this studyincluded two that were made against MN-rgp120, 1026 and 13H8, which weresequence dependent and recognized the V3 and C4 domains respectively(54, 55). In addition, we included the broadly neutralizing, CD4blocking MAb b12 (10, 57), as well as CD4-IgG (11), both of which bindto conformation dependent sites involving several regions of themolecule.

Using a standard ELISA, we compared antibody binding to cathepsin Ltreated and untreated MN-rgp120. The digestions ran to completion asjudged by the absence of intact gp120 when resolved by polyacrylamidegel electrophoresis. We found that cathepsin L digestion of gp120destroyed the ability to bind both the V3 domain specific, virusneutralizing 1026 MAb, and the C4 domain specific, CD4-blocking 13H8MAb. Much of the binding to b12 and CD4 IgG was preserved by cathepsin Ldigestion; however, there was a significant reduction in bindingaffinity. This result can be explained by the fact that the two C4sites, G431-K432 and Y435-A436, and one V3 site, K327-G328, are locatedin close proximity to the epitopes recognized by the 13H8 and 1026 MAbs.A different pattern of binding was observed with cathepsin D treatedgp120. In these experiments, the binding to 13H8 and 1026 was preserved,although there appeared to be some reduction in binding affinity of the1026 MAb. In addition, there was a large reduction in binding to b12 aswell as to CD4-IgG. The inability of cathepsin D treatment to inhibitthe binding of the 13H8 MAb can be attributed to the fact that thecathepsin D cleavage sites are located in the V2 and C2 domains, andremote from the conformation independent 13H8 epitope in the C4 domain.The large decrease in binding affinity of the b12 MAb and CD4-IgG tocathepsin D treated gp120 might be explained by the fact that sequencesin the C2 domain are known to be important for maintaining the structureof the CD4 binding site, and that binding of the b12 MAb is dependent oncontact sites in this region (46, 88). Together these resultsdemonstrate that cathepsin cleavage sites are located in regions ofgp120 recognized by neutralizing MAbs and CD4-IgG, and that cleavage bycathepsins L and D differentially alters antibody and CD4 binding tothese sites.

Discussion

In these studies we have identified the location of cleavage sites onMN-rgp120 recognized by three proteases (cathepsin L, cathepsin S andcathepsin D) thought to be important in antigen processing andpresentation. We found that these sites are not randomly distributed,but rather occurred in regions of the envelope glycoprotein known topossess receptor binding and attachment sites and epitopes recognized byneutralizing antibodies. Comparative sequence analysis showed that manyof these sites are highly conserved in the major clades of HIV with somebeing conserved in both the chimpanzee form of HIV as well as SIV.Finally we showed that cleavage by cathepsins L and D diminished thebinding of neutralizing antibodies and CD4-IgG. We found that none ofthe experimentally determined cathepsin cleavage sites matched thecathepsin cleavage sites predicted by enzyme cleavage site predictionprograms (Table 1).

To some extent, the ability to predict cathepsin cleavage sites has beenlimited by the availability of experimental data as indicated in theMEROPS Peptidase Database (Rawlings et al. 2008). Moreover there isuncertainty as to the extent to which cathepsin recognition sequencesextend upstream and downstream of the cleavage site. The listing ofN-terminal and C-terminal flanking sequences for the sites defined inthis study is provided in supplemental information Tables S1 and S2 andwill contribute to our knowledge of cathepsin recognition motifs.

Remarkably seven of the cathepsin cleavage sites identified in thisstudy were located in regions of the envelope protein known to beassociated with receptor binding or the binding of neutralizingmonoclonal antibodies. For example, the V2 domain is known to containepitopes recognized by virus neutralizing antibodies and has been termedthe global regulator of virus neutralization. Moreover the L181-Y182cathepsin D cleavage sites are located just one amino acid away from thealpha-4-beta-7 receptor binding site (LDI/V) recently reported by Arthoset al. The V3 domain is known as the principal neutralizing determinantand contains epitopes recognized by a variety of neutralizing antibodiesand is a key determinant of chemokine receptor tropism. The C4 domain isknown to possess multiple contact residues for CD4 binding, chemokinereceptor binding, and the binding of CD4 blocking, neutralizingantibodies. The importance of the CD4 binding site in antigen processingwas noted by Tuen et al. (2005) who reported that antibodies to the CD4binding site inhibited cleavage by antigen processing enzymes andsubsequent MHC class II antigen presentation. Sequences in the C2 domainhave been reported to be important for both CD4 binding and chemokinereceptor binding, and it is remarkable that one of the cathepsin S sitesidentified in the C2 domain is located at a CD4 contact residue and theother is located at a chemokine receptor contact residue. It isdifficult to understand how this remarkable correspondence betweenreceptor binding sites and cathepsin cleavage sites could occur bychance. This is particularly significant in view of the fact that thereare several domains in gp120 that appear to be devoid of cathepsincleavage sites. These include the C1, V1, V4, and V5 domains which lackcathepsin L cleavage sites. However our data suggest that one or morecathepsin S and cathepsin D cleavage sites remain to be located betweenthe N-terminus and the V2 domain.

The functional importance of the cathepsin cleavage sites identifiedabove was further supported by the observation that six of the eightcathepsin cleavage sites were highly conserved in HIV, with one,G431-K432 in the C4 domain, being conserved in HIVcpz as well as SIV.Previous studies have suggested that many viruses, including, HIV, haveevolved mechanisms to alter antigen processing as a way to escape ordirect the immune response to their advantage, see Wolf, P. 1995 AnnuRev Cell Dev Biol 11:267-306. Most of these mechanisms affect MHC classI restricted cellular immune responses; however, mechanisms that alterMHC class II antigen presentation have also been reported (Keele, B. F.2008. Proc Natl Acad Sci USA 105:7552-7.). HIV has developed a varietyof mechanisms to evade the immune response. HIV directly destroys CD4+helper T cells required for effective control of virus replication, anda lack of effective T-cell help is thought to limit the antiviral immuneresponse. Other mechanisms to evade the immune response include the highlevel of sequence variation that is evident in all HIV proteins, butparticularly evident in the envelope protein that incorporates manyinsertions and deletions. The virus also appears to have evolved epitopeconcealment mechanisms in the envelope protein that restrict access toantibody binding at neutralizing sites in the V3 domain, CD4 bindingsite, and membrane proximal external region (MPER). Finally, the largenumber of N-linked glycosylation sites on gp120 which are thought toform a protective “glycan shield” that provides yet another level ofprotection from the binding of neutralizing antibodies.

The results of our studies suggest that HIV may have evolved anothermechanism of immune escape involving incorporation of protease cleavagesites in regions important for receptor binding and the binding ofneutralizing antibodies. Cleavage at these sites may direct or modulatethe immune response in such a way as to prevent the formation ofneutralizing antibodies or prevent recognition of existing neutralizingantibodies. Our results suggest that the cleavage sites recognized byenzymes important for MHC class II antigen processing are highlyconserved and localized to functionally specific regions of the envelopeglycoprotein. Because of the extraordinarily high level of sequencevariation in HIV-1, resulting from high mutation and replication ratesas well as immune selection, it is unlikely that these sites could bepreserved unless they provided a significant fitness advantage for thevirus.

Recent studies by Tenzer et al. (Virology 372:273-90) suggested that theimmunodominance of CTL epitopes is determined by proteosome digestionprofiles and trimming by endoplasmic reticulum aminopeptidases. Theyfurther showed that CTL escape mutations involved amino acidsubstitutions that affected proteosome cleavages directly or sequencesflanking cleavage sites in p17 and p24. The results from the presentstudies are consistent with the possibility that HIV might similarlyregulate the immunodominance of MHC class II restricted immune responsesby tightly controlling proteolysis by the enzymes required for MHC classII antigen processing. The observation that the antigen processing sitesare highly conserved is itself remarkable and consistent with thishypothesis. The additional observation that these sites are located inregions associated with receptor binding and neutralizing antibodiesbinding is especially noteworthy and suggests important functionalsignificance. It should be emphasized that while Tenzer at al. suggeststhat protease cleavage affects the immunogenicity of the cytotoxiclymphocyte immune response to HIV core proteins, the present work issignificantly novel in that we have discovered that protease cleavageaffects the immunogenicity of antibody mediated immune response.

One potential explanation for the conservation of cathepsin cleavagesites at receptor binding sites is the fact that the receptor bindingsites are among the few sites on the virion associated envelope proteinsthat are not protected by the protective glycan shield and thus may bethe only sites accessible to proteases. However, it is unlikely thatthis can explain the data since gp120 is readily shed from viruses andmonomeric gp120 has multiple exposed regions that are not glycosylated.An alternative explanation may relate to an additional immune escapemechanism first described for poliovirus.

Studies with poliovirus type 3 have shown that a major neutralizingepitope (antigenic site 1) contains a protease cleavage site, and thatcleavage at this site prevents the binding of neutralizing antibodies.The authors suggested that this protease site may have evolved as ameans by which the virus could escape from neutralizing antibodiesdirected to this site. The incorporation of protease cleavage sites atneutralizing epitopes, in effect, causes neutralizing epitopes to “selfdestruct” after coming into contact with serum or cellular proteases.The possibility that epitopes recognized by neutralizing antibodies arelabile and subject to destruction by extracellular proteases before theycan stimulate antigen receptors on B cells is intriguing and couldexplain why it has been so difficult to elicit neutralizing antibodieswith recombinant envelope proteins, despite the fact that they clearlypossess the capacity to absorb broadly neutralizing antibodies from HIV+sera. The effect of such cleavage could be to prevent the formation ofneutralizing antibodies to the intact virus envelope protein or theprevention of existing neutralizing antibodies to important neutralizingepitopes. For this type of mechanism to be operative, one would need toshow that cathepsin proteolysis is able to destroy the epitopesrecognized by neutralizing antibodies, and that cleavage would need tooccur prior to exposure of gp120 to antigen receptors on B cells. Theantibody binding studies described in this paper showed that the bindingof neutralizing antibodies and CD4-IgG was significantly reduced, and insome cases completely prevented, by cathepsin cleavage. These results inpart fulfill the first requirement of this epitope “self-destruct”hypothesis. However, these cathepsins are best known as lysosomal andendosomal enzymes and therefore, would not be expected to come in directcontact with HIV virions. Examination of the literature revealed thatseveral cathepsins (e.g. cathepsins L, B, S, and K) can be secreted andare known to play an important role in cancer biology, tissueremodeling, and inflammatory diseases (15, 48, 56, 86). The release ofthese enzymes has not been studied in the course of HIV infection;however, cathepsin S has been reported to be secreted from activatedmacrophages (63). While proteolysis of virion-associated envelopeproteins would be expected to inhibit virus infectivity, it is doubtfulthat this cleavage would be 100% effective. The high levels of plasmaviremia and integrated provirus that occur in HIV infection would likelyinsure that infection is sustained even if a large percentage of virusis inactivated by protease cleavage. Since our studies show that gp120is highly sensitive to cathepsin S, and because cathepsin S is unique inbeing highly active at neutral pH, and because cathepsin S sites arelocated in close proximity to neutralizing sites in the C2, V3, and C4domains, this enzyme is a logical candidate to mediate epitopedestruction in vivo.

While the role of cathepsins on the MHC class II immune responses isundisputed, they may also play an important role in MHC class Iresponses to HIV. A variety of MHC class I restricted CTL epitopes occurat or in close proximity to the cathepsin cleavage sites identified inthis paper. These include the cathepsin S site in the C2 domain, thecathepsin S and L sites in the V3 domain, and the cathepsin L sites inthe C4 domain. The co-location of these CTL epitopes with the cathepsincleavage sites identified in this paper may result from the TAPindependent “crosspresentation” pathway that has been documented fordendritic cells and macro phages and known to require cathepsin S. Thispathway enables proteosome independent MHC class I restrictedpresentation of peptides generated by cathepsin S cleavage.Identification of antigen processing sites promises to provide a newunderstanding of the molecular basis of the specificity of the immuneresponse to HIV envelope glycoprotein. Insertion or deletion ofcathepsin cleavage sites may provide a new approach to refocus bothhumoral and cellular antiviral immune responses. Studies to explore thispossibility are in progress. Proteases are estimated to represent ˜2% ofthe genes in the human genome (62) and it would not be surprising thatHIV has evolved additional strategies to use proteases to its advantage.The studies described will contribute to our understanding of thespecificity of antiviral immune responses and will add to our knowledgeof the role of proteases in HIV biology.

Tables from SC-2010-117

Sequence ID  Protein or  number protein segment Species * SEQ ID NO: 7GlyThrIleArgGln HIV 1 SEQ ID NO: 8 LysAlaMetTyr HIV-1 SEQ ID NO: 9AlaProProIle HIV-1 SEQ ID NO: 10 AlaCysProLys HIV-1 SEQ ID NO: 11ThrGlnLeuLeu HIV-1 SEQ ID NO: 12 ThrLysAsnIle HIV-1 SEQ ID NO: 13TyrLysLeuAsp HIV-1 SEQ ID NO: 14 ValValIleArgSer HIV-1 SEQ ID NO: 15LysTyrAlaLeu HIV-1 SEQ ID NO: 22 MN HIV-1 SEQ ID NO: 23 HXB2 HIV-1SEQ ID NO: 24 A1.KE.94.Q23_17 HIV-1 SEQ ID NO: 25 A1.UG.92.92UG037 HIV-1SEQ ID NO: 26 C.ET.86.ETH2220 HIV-1 SEQ ID NO: 27 C.IN.93.93IN101 HIV-1SEQ ID NO: 28 D.TZ.01.A280 HIV-1 SEQ ID NO: 29 D.UG.94.94UG114 HIV-1SEQ ID NO: 30 AE.TH.93.93TH051 HIV-1 SEQ ID NO: 31 AE.TH.90.CM240 HIV-1SEQ ID NO: 32 CPZ.CM.05.SIVcpzMT145 SIV SEQ ID NO: 33 CPZ.US.85.CPZUSSIV SEQ ID NO: 34 SIV.US.MAC251 SIV SEQ ID NO: 35 SIV.US.MAC239 SIV *HIV-1: Viruses; Retro-transcribing viruses; Retroviridae;Orthoretrovirinae; Lentivirus; Primate lentivirus group SIV: Viruses;Retro-transcribing viruses; Retroviridae; Orthoretrovirinae; Lentivirus;Primate lentivirus group

TABLE 1 Identification of cathepsin cleavage sites on MN-rgp120 identifiedby N-terminal sequencing Cleavage N-terminal Associated Proteases Sizesite sequence Domain function* Sequence No. Cathepsin L  70 k A₃-L₄ LADHSV-1 gD Flag  50 k K₃₂₇-G₃₂₈ GTIRQ V3 3 SEQ ID NO: 7  45 k K₃₂₇-G₃₂₈GTIRQ V3 3 SEQ ID NO: 7  35 k K₃₂₇-G₃₂₈ GTIRQ V3 3 SEQ ID NO: 7  20 kG₄₃₁-K₄₃₂ KAMY C4 1, 2, 4 SEQ ID NO: 8  14 k Y₄₃₅-A₄₃₆ APPI C4 2, 4SEQ ID NO: 9 Cathepsin S  60 k  Q₂₀₈-A₂₀₉ ACPK C2 1, 2 SEQ ID NO: 10 50 k S₂₆₁-T₂₆₂ TOLL C2 3 SEQ ID NO: 11  38 k T₃₂₂-T₃₂₃  TKNI V3 3SEQ ID NO: 12  18 k Y₄₃₅-A₄₃₆ APPI C4 2, 4 SEQ,ID NO: 9  12 k Y₄₃₅-A₄₃₆APPI C4 2, 4 SEQ ID NO: 9 3.5 k — — — Cathepsin D  70 k L₁₈₁-Y₁₈₂ YKLDV2 3, 5 SEQ ID NO: 13  55 k E₂₇₄-V₂₇₅ VVIRS C2 N/A SEQ ID NO: 14  52 kE₂₇₄-V₂₇₅ VVIRS C2 N/A SEQ ID NO: 14  45 k — — —  30 k E₂₇₄-V₂₇₅ VVIRSC2 N/A SEQ ID NO: 14  20 k L₁₈₁-Y₁₈₂  YKLD V2 4, 5 SEQ ID NO: 13  12 kE₂₇₄-V₂₇₅ VVIRS C2 N/A SEQ ID NO: 14  10 k — — —   6 k — — —   5 k —KYAL HSV-1 gD Flag SEQ ID NO: 15   4 k — KYAL HSV-1 gD FlagSEQ ID NO: 15 *1, indicates CD4 binding site; 2, indicates chemokinereceptor binding site; 3, indicates V3 domain neutralizing antibodybinding site; 4, indicates b12 antibody binding site; 5, indicates α4β7binding site; N/A, indicates data not available; and Flag, indicatessequence from herpes simplex virus glycoprotein D used as a flag epitopeto facilitate purification.

TABLE 2 Conservation of cathepsin cleavage sites in HIV sequencedatasets* Polymorphism prevalence in HIV cohorts (%) Observed cleavagesites for MN VAX004 Keele Los Alamos Domain Cathepsin Location Site n =1047 n = 2908 n = 1766 V2 D 181-182 LY FY (81.4) FY (81.0) FY (92.2) C2S 208-209 QA QA (97.6) QA (96.8) QA (97.3) C2 S 261-262 ST ST (97.4) ST(99.3) ST (97.9) C2 D 274-275 EV EV (61.3) EV (60.6) EI (39.8) V3 S322-323 TT AT (59.4) AT (49.5) AT (58.0) V3 L 327-328 KG IG (87.1) IG(91.6) IG (83.2) C4 L 431-432 GK GK (91.5) GK (96.3) GK (36.6) C4 S, L435-436 YA YA (98.9) YA (97.7) YA (97.7) *The VAX004 dataset of clade Bviruses from the US was obtained from the GSID HIV Sequence database(www.gsid.org); a dataset of clade B viruses from acute infections(Keele et al., 2008) and a dataset of world-wide isolates of HIV wereobtained from the Los Alamos HIV Sequence database (www.lanl.hiv.gov).

SUPPLEMENTAL TABLE S1 Predicted cathepsin cleavage sites in MN-rgp120Cleavage Cathepsin Site P4 P3 P2 P1 P1′ P2′ P3′ P4′ L 45-46 P V W K E AT T 96-97 N M W K N N M V 183-184 L L Y K L D I E 197-198 T S Y R L I SC 471-472 E I F R P G G G 482-483 D N W R S E L Y 487-488 E L Y K Y K VV 489-490 Y K Y K V V T I S 354-355 S K L K E Q F K 421-422 C K I K Q II N 440-441 P P I E G Q I R D 215-216 K I S F E P I P Predictedcathepsin L, S and D cleavage sites in MN-rgp120. The location ofcathepsin cleavage sites and flanking sequences was predicted using themethod of Boyd et al. (6) and cleavage specificity algorithms forcathepsin L and S from Choe et al. (17) and cathepsin D from Scarboroughet al. (73). The scissille bond, located between the P1 and P1′residues, and the flanking residues are listed according to thenomenclature of Schechter and Berger (74).

SUPPLEMENTAL TABLE S2 Observed cathepsin cleavage sites in MN-rgp120Cathepsin Site P4 P3 P2 P1 P1′ P2′ P3′ P4′ L 327-328 K N I K G T I R431-432 Q K V G K A M Y 435-436 K A M Y A P P I S 208-209 V I T Q A C PK 261-262 P V V S T Q L L 322-323 A F Y T T K N I 435-436 K A M Y A P PI D 181-182 Y A L L Y K L D 274-275 A E E E V V I R Experimentallydetermined cathepsin L, S and D cleavage sites in MN-rgp120. Thelocation of cathepsin cleavage sites and flanking sequences wasdetermined for MN-rgp120 by Edman sequence degradation of peptidesrecovered after protease digestion (see Material and Methods). Thescissille bond, located between the P1 and P1′ residues, and theflanking residues are listed according to the nomenclature of Schechterand Berger (74).Tables from SC2009-449

TABLE 1 Neutralization in 108059 and 108060 108059 Wild Type Viruses108060 Wild Type Viruses A Sera/Neutralization Titers* BSera/Neutralization Titers Clone Z1679 Z1684 N16 Z23 Clone Z1679 Z1684N16 Z23 002 <40 <40 <40 251 022 53 58 51 117 005 <40 <40 <40 234 024 804609 612 1667 008 <40 <40 <40 244 002 303 160 195 379 010 <40 <40 <40 238003 69 57 67 151 013 <40 <40 <40 196 011 136 130 177 222 014 <40 <40 <40436 012 62 57 70 241 016 44 50 49 490 013 53 50 58 158 018 <40 <40 <40167 018 428 243 388 1378 021 <40 <40 <40 278 019 44 <40 40 145 023 <40<40 <40 258 021 47 47 70 157 The neutralizing antibody titer (IC50) isdefined as the reciprocal of the plasma dilution that produces a 50%inhibition in target cell infection. Values in bold representneutralization titers that are at least 3 times greater than thoseobserved against the negative control (aMLV). All clones tested wereCCR5 tropic. Clone indicates gp160 envelope genes.

TABLE 2 Neutralization of Wild Type (WT) and Mutated Clones from Subject108060 by HIV+ sera possessing broadly neutralizing antibodies Mutationof Clone 022 A wtR from 108060 Mutation at Position 655 Clone/Sera/Neutralization Titers* B Sera/Neutralization Titers* Mutants Z1679Z1684 N16 Z23 Clone mutant Z1679 Z1684 N16 Z23 022 wtR 75 104 76 384 022wtR 40 <20 36 281 024 wtS 728 1086 982 1926 024 wtS 1099 1193 545 4167N323S 73 95 54 382 022 Q655R 14276 2876 2610 8422 N530G 37 42 41 308 022Q655K 5486 8590 4276 19476 K634E 67 73 72 346 022 Q655E 564 132 366 2424Q655R 2165 2562 4472 8290 022 Q655S 1565 472 674 2650 I827T 39 <20 113<100 022 Q655N 148 24 57 820 832/833 104 50 63 404 022 I827T 49 <20 <20277 827/832/833 72 53 81 279 024 R655Q 50 <20 39 372 The neutralizingantibody titer (IC50) is defined as the reciprocal of the plasmadilution that produces a 50% inhibition in target cell infection. Valuesin bold represent neutralization titers that are significantly above thebackground (Experimental Procedures). All clones tested were CCR5tropic. Clone indicates gp160 envelope genes. wtR and wtS indicate wildtype neutralization-resistant and -sensitive clones respectively.

TABLE 3 Transfer of Q655R Mutation to Unrelated Viruses: Sensitivity toNeutralizing Monoclonal Antibodies and Entry Inhibitors IC50 MAbs andFusion Inhibitors (μg/ml) Clone Mutation 2F5 4E10 b12 2G12 Fuzeon CD4IgG 108060_022 wtR 3.250 5.201 >20 >20 0.068 >20 108060_022 Q655R 0.0930.156 >20 >20 0.004 0.161 108060_024 wtS 0.151 0.333 >20 >20 0.019 0.798108060_024 R655Q 3.434 6.546 >20 >20 0.130 >20 108069_005 wtR 1.1293.556 >20 >20 0.071 >20 108069_011 wtS 0.043 0.040 >20 >20 0.145 >20108069_005 Q655R* 0.052 0.044 >20 >20 0.011 1.080 108051_005wtR >20 >20 >20 >20 0.088 >20 108051_006 wtS 1.176 1.369 >20 >20 0.0080.231 108051_005 Q655R* 0.343 1.314 >20 >20 0.036 5.209 *Numbering withreference to 108060 protein. The neutralizing antibody titer (IC50) isdefined as the concentration (μg/ml) of mAB or entry inhibitor thatproduces a 50% inhibition in target cell infection. Values in boldrepresent neutralization titers that are significantly above thebackground (Experimental Procedures). All clones tested were CCR5tropic. Clone indicates gp160 envelope genes. wtR and wtS indicate wildtype neutralization-resistant and -sensitive clones respectively.

TABLE 4 Sensitivity to neutralizing monoclonal antibodies and entryinhibitors in 108060 clones and unrelated viruses (a) IC₅₀ (μg/ml) ofindicated MAb or fusion inhibitor Clone Mutation 2F5 4E10 b12 2G12Enfuvirtide CD4-IgG 108060_022 wtR 3.250 5.201 >20 >20 0.068 >20108060_022 Q655R 0.093 0.156 >20 >20 0.004 0.161 108060_024 wtS 0.1510.333 >20 >20 0.019 0.798 108060_024 R655Q 3.434 6.546 >20 >20 0.130 >20108069_005 wtR 1.129 3.556 >20 >20 0.071 >20 108069_011 wtS 0.0430.040 >20 >20 0.145 >20 108069_005 Q655R^(b) 0.052 0.044 >20 >20 0.0111.080 108051_005 wtR >20 >20 >20 >20 0.088 >20 108051_006 wtS 1.1761.369 >20 >20 0.008 0.231 108051_005 Q655R^(b) 0.343 1.314 >20 >20 0.0365.209 (a) The neutralizing antibody titer (IC₅₀) is defined as theconcentration (μg/ml) of an MAb or entry inhibitor that produces a 50%inhibition in target cell infection. Values in bold representneutralization titers that are significantly above the background (seeMaterials and Methods). All clones tested were CCR5 tropic. Clonesindicate gp160 envelope proteins, wtR and wtS indicate wild-typeneutralization-resistant and -sensitive clones, respectively.^(b)Numbering with reference to subject 108060 protein.

The invention claimed is:
 1. A formulation comprising an HIV envelope glycoprotein polypeptide comprising a single amino acid substitution in a helix of the HIV envelope glycoprotein polypeptide, wherein the helix is selected from the group consisting of the N36 helix and the C34 helix, wherein said single amino acid substitution increases the ability of said polypeptide to bind neutralizing antibodies, wherein the single amino acid substitution is selected from the group consisting of V551[*], Q553[*], and Q655[*], where [*] represents any amino acid other than Q or N, and wherein the numbering of the substituted amino acid is with reference to the amino acid sequence set forth in SEQ ID NO:
 16. 2. The formulation of claim 1, comprising an excipient, carrier or adjuvant.
 3. The formulation of claim 1, wherein the single amino acid substitution is V551[*], where [*] represents any amino acid other than Q or N.
 4. The formulation of claim 1, wherein the single amino acid substitution is Q553[*], where [*] represents any amino acid other than Q or N.
 5. The formulation of claim 1, wherein the single amino acid substitution is Q655[*], where [*] represents any amino acid other than Q or N.
 6. The formulation of claim 5, wherein the single amino acid substitution is Q655R.
 7. The formulation of claim 5, wherein the polypeptide comprises the sequence set forth in SEQ ID NO:
 16. 8. The formulation of claim 3, comprising an excipient, a carrier or an adjuvant.
 9. The formulation of claim 4, comprising an excipient, a carrier or an adjuvant.
 10. The formulation of claim 5, comprising an excipient, a carrier or an adjuvant.
 11. The formulation of claim 6, comprising an excipient, a carrier or an adjuvant.
 12. The formulation of claim 7, comprising an excipient, a carrier or an adjuvant. 